Photolabile esters and their uses

ABSTRACT

Compounds which are capable of generating acid on photolysis are disclosed, and the uses of these compounds, especially for deprotecting the termini of nucleic acid molecules or peptides during synthesis of arrays. The compounds described herein may be employed in the detritylation of 5′-O-dimethoxytrityl (DMT) protected nucleotides by photolysing the compounds to generate an acid capable of removing the DMT group allowing oligonucleotide arrays to be synthesised using readily available 5′-O-DMT-nucleoside-3′-O-phosphoramidite monomers conventionally used in solid phase nucleic acid synthesis. A method of avoiding the effects of stray light in projection lithography techniques is also disclosed.

FIELD OF THE INVENTION

The present invention relates to photolabile esters and more especiallyto photolabile esters which generate acid on photolysis. The presentinvention further relates to the uses of these compounds, in particularfor removing protecting groups in the synthesis of oligonucleotides orpeptides, e.g. in the construction of microarrays.

BACKGROUND OF THE INVENTION

Photolithography and direct photochemical unblocking are techniques usedin microarray technology to build up arrays of oligonucleotides orpeptides binding agents at defined locations on solid support. Althoughthis approach is employed to generate arrays for use in some analyticalapplications based on DNA-hybridisation, the high cost ofphotolithographic masks, the use of non-commercial photolabilephosphoramidite monomers and problems with quantitative, directphotochemical unblocking lead to very expensive and often poor qualitymicroarrays³⁰.

One attempt to solve these problems is based on so-called chemicalamplification. This technique is borrowed from the electronics industry,and uses a combination of photochemically and thermally generated acidproduction at desired sites on an array surface covered with adiffusion-limiting polymeric film³¹. However, the need to use strongacids such as benzenesulphonic acid in the key photodirected step, whileacceptable in the production of intergrated circuits, makes applicationof this approach with acid labile purine nucleosides difficult^(32,33).There have been attempts to overcome these problems by designing newphotoacid generating compounds, but on the whole, these attempts havenot been successful. By way of example, Le Proust et al²⁸ and Gao etal²⁷ have employed photolabile hexafluoroantimonates in the synthesis ofoligonucleotides in solution (see also WO99/41007). However, thesephotoacid generators involve the release of free radicals which canresult in undesirable side reactions such as blockage of 5′-OH group.

Reichmannis et al³⁴ described 2-nitrobenzyl esters which photolyse toproduce trimethylacetic acid, and more particularly the mechanism ofthis reaction and its yield, both in solution and in a polymer matrix.WO00/66259 describes the use of photoactivated reagents which whenactivated are capable of removing protecting groups at the termini ofsubstrates being synthesised on a solid phase. The application suggeststhe use of triarylsulphonium hexafluorantimonates, triarylsulphoniumhexafluorophosphates, 2,1,4-diazonapthoquinone sulphonates andperhalogenated traiazines. In one example,1-[2-nitrophenyl]ethyl-1-trichloroacetate is irradiated with UV light togenerate trichloroacetic acid for removing protecting 5′-dimethoxytritylgroups from oligonucleotide substrates.

The actual fabrication of arrays of binding agents and in particularoligonucleotide or peptide arrays is an area of intense interest in theart. The chemical synthesis of binding agents such as oligonucleotidesis well known. The most commonly used method is a solid phase synthesisusing controlled porosity glass or equivalent material as the support.Stepwise extension of an oligonucleotide attached via a linker moleculeto the support occurs by addition of one nucleotide at a time.Attachment to the linker is commonly through the oligonucleotide-3′-OH,with chain extension therefore at the oligonucleotide-5′-OH. Because ofthe stepwise nature of the process, satisfactory synthesis ofoligonucleotides of commonly desired chain lengths of 20 or morenucleotides requires a high stepwise yield. The overall yield of anN-mer synthesised with a stepwise yield of Y is Y^(N), and diminishesrapidly once Y falls beneath about 0.95. After completion of synthesisthe oligonucleotide is cleaved from the support and purified prior touse. Caruthers³⁵ and Beaucage & Iyer³⁶ have written detailed reviews ofoligonucleotide synthesis methods.

Typically, each new oligonucleotide monomer is added to a growingoligonucleotide as a modified nucleotide substituted at its 5′-OHposition with a 4,4′-dimethoxytrityl group, and abeta-cyanoethyl-phosphoramidite at its 3′-OH position. Synthesis startsby coupling the first nucleotide through its 3′-OH group to the terminalhydroxyl group of a linker molecule attached to a solid support. Anyunreacted terminal OH groups are then blocked with acetic anhydride, andthe trivalent phosphite is oxidised to pentavalent phosphate. Thedimethoxytrityl group is then removed with acid from the 5′-OH positionof the first nucleotide, which can then react with the next nucleotidephosphoramidite to be added. The cycle of steps is then repeated untilthe desired chain length has been synthesised. Finally, alkali treatmentis used to remove N-protective groups and also to cleave analkali-labile bond in the linker, thereby releasing the oligonucleotidewhich may then be purified.

In prior art solid phase synthesis methods, removal of DMT, ordetritylation, is effected with di- or trichloroacetic acid according toequation (1). Stronger acids cause chain breakage by depurination. Itshould be noted that protons are reagents, not catalysts, in thedetritylation reaction, and are consumed with a stoichiometry of 1proton per DMT⁺ cation released, as in equation (1):oligonucleotide-5′-O-DMT+H ⁺=oligognucleotide-5′-OH+DMT ⁺  (1)

The repetitive cycle of steps in the synthesis of oligonucleotide, orindeed peptide, lends itself to automation, and a variety ofcommercially available instruments have been developed for that purpose.The availability of synthetic oligonucleotides has led to thedevelopment of arrays of oligonucleotides on paper or other polymericsheets, fabric or glass, allowing multiple hybridisation reactions to becarried out in parallel.

Early descriptions of oligonucleotide arrays were from academiclaboratories and the array densities achieved were modest. Constructionmethods define three classes of array, namely (a) arrays printed frompre-synthesised oligonucleotides, (b) arrays synthesised in situ byreagent printing and (c) arrays synthesised in situ by a photodirectedmethod.

The printing methods create array elements at a modest density, up to5,000/cm², with each element having a diameter of about 0.1 mm andseparated from its neighbours by a similar distance and possibly anadditional physical barrier to prevent reagents that should beconstrained to selected elements from spreading to adjacent elements.Photolithographic methods currently achieve much higher densities(160,000/cm²), with the potential for even higher (10⁶/cm²).

Photodirected synthesis of oligonucleotides in arrays was firstdescribed in 1991 by Fodor et al³⁷. The main technical innovation was toreplace the conventional acid-removable dimethoxytrityl blocking groupat the oligonucleotide 5′-OH with a group that was photo-removable. Thearray elements at which groups would be unprotected, and thereforereactive with whichever A, C, G orT-deoxyribonucleotide-3′-O-phosphoramidite was subsequently applied,were determined by patterned illumination of the array surface.Proximity or contact photolithography is needed to minimise stray light,and requires numerous high precision physical masks (metal on glass orquartz), with the result that this technology has the significantdisadvantages of high cost and low flexibility.

In the photodirected method for making arrays, the synthesis consists ofa cycle of steps that adds a nucleotide at each chain length tophotoselected array elements. The cycle is used four times (once eachfor A, C, G & T) to extend the length of the array by one nucleotide,and 4N times to make an array of N-mers. The first four cycles couplemonomer to a linker attached to the glass or other solid surface. Thelinker has an aliphatic —OH group at its free end. All subsequent cyclescouple monomer to oligonucleotide 5′-OH. The sequence of actions in thephotodirected synthesis of an oligonucleotide array, using nucleotidemonomers with photolabile protection of the 5′-OH group is given inTable 1.

The use of contact or close proximity photolithography uses masks ofmetal on glass or quartz. The transmission of light through themetallised areas of the mask is 10⁻⁵ of that transmitted through theclear (non-metallised) areas (Pease et al²⁹). In other words, thecontrast ratio of metal on glass masks is 10⁵. The associated intensitylevel of stray light is negligible in the context of photodirectedsynthesis of oligonucleotide arrays. However, the masks are expensive,and the number needed is large (100 for a 25-mer array), making thismethod of fabricating arrays unsuitable for use outside an expensivelyequipped industrial environment.

To overcome this drawback of expense and inflexibility, several groups(Singh-Gasson et al³⁸, Garner³⁹ and Staehler⁴⁰) have reported the use ofprojection photolithography using Digital Micromirror Device (DMD:Hornbeck⁴¹) projectors) in association with photosensitive blockinggroups of the oligonucleotide-5′-OH group. The aim was to avoid the costand inflexibility of metal-on-glass physical masks by replacing themwith programmable masks in silico that determine the patterned output ofa light projector. LeProust et al²⁸ have also used a DMD projector, butused photoacid generation to deprotect the tritylatedoligonucleotide-5′-OH group.

Furthermore, despite the work described above, it remains a problem inthe art in generating acid for in situ deprotection of oligonucleotides.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to compounds which are capable ofgenerating acid on photolysis, and to the uses of these compounds,especially for deprotecting the termini of nucleic acid molecules orpeptides during synthesis. Thus, for example, the compounds describedherein can be employed in the detritylation of 5′-O-dimethoxytrityl(DMT) protected nucleotides by photolysing the compounds to generate anacid capable of removing the DMT group. This means that the presentinvention has the significant advantage of permitting chemistry to beused for the synthesis of oligonucleotide arrays which employscommercially available 5′-O-DMT-nucleoside-3′-O-phosphoramidite monomersconventionally used in solid phase nucleic acid synthesis^(35,36), butcritically adapts this robust chemistry so that it employs a photoacidgeneration to effect detritylation reactions at specific locations(elements) of an array of locations on a surface.

Further, unlike many prior art approaches for synthesising microarrays,the acids generated by photolysis of the compounds of the inventionbalance the requirements of being sufficiently strong to ensure that thedeprotection step proceeds in a rapid manner, e.g. the reaction requiresonly thirty seconds or so, and does not lead to significant degradationof acid labile purine nucleosides. Preferred compounds of the inventionalso have the advantage that they have satisfactory extinctioncoefficients, e.g. 3500 cm.M⁻¹, undergo efficient photolysis, e.g. witha quantum yield of c. 0.4, and lead to only one UV absorbing productwhen analysed by HPLC and TLC. Further, the work described hereindemonstrates quantitative production of acid (H⁺) and that the compoundswere stable and easy to work with provided that simple precautions weretaken to avoid ambient UV light.

The work also predicts errors in oligonucleotide array synthesis causedby stray light at levels characteristic of maskless projection³⁸⁻⁴¹using computer controlled DMA's, and identifies a method of amelioratingor preventing such errors in those cases where photodirected synthesisis provided by photoacid or photobase generation.

Accordingly, in a first aspect, the present invention provides acompound represented by the formula:

wherein:

-   -   R¹ is selected from hydrogen, aryl or substituted aryl, aryloxy        or substituted aryloxy, or an unsubstituted or substituted        heterocyclic group;    -   R² is selected from hydrogen, halogen, alkyl or substituted        alkyl, alkoxy or substituted alkoxy, aryl or substituted aryl,        aryloxy or substituted aryloxy, amino or substituted amino, or a        nitro group;    -   R³ is selected from hydrogen, alkoxy or substituted alkoxy, aryl        or substituted aryl, aryloxy or substituted aryloxy, amino or        substituted amino, or an unsubstituted or substituted        heterocyclic group;    -   R⁴ is an alkyl group substituted with one or more halogen        substituents, such as ClCH₂, Cl₂CH, Cl₃C or F₃C;    -   R⁵ is selected from hydrogen, halogen, alkyl or substituted        alkyl, alkoxy or substituted alkoxy, aryl or substituted aryl,        aryloxy or substituted aryloxy, amino or substituted amino, a        nitro group or an unsubstituted or substituted heterocyclic        group; and,    -   R⁶ is selected from hydrogen, halogen, alkyl or substituted        alkyl, alkoxy or substituted alkoxy, aryl or substituted aryl,        aryloxy or substituted aryloxy, or amino or substituted amino,        or an unsubstituted or substituted heterocyclic group.

In the above definitions, preferred substituents are C₁₋₁₀, and morepreferably C₁₋₅, and may be straight chain, branched, cyclic orheterocyclic. Examples of groups that may be included in substitutedfunctional groups, as defined above, include halogen (F, Cl, Br or I),alkyl or substituted alkyl, alkoxy or substituted alkoxy, aryl (Ar) orsubstituted aryl, N₂, CN, COOR, where R is hydrogen, halogen or an alkylgroup, and unsubstituted or substituted heterocyclic groups.Heterocyclic groups include those with one or more heteroatoms (e.g. N,O or S) in a saturated, unsaturated or aryl ring system, e.g. aheterocyclic group may comprise one or two nitrogen atom in a five orsix membered ring or one oxygen atom in a five or six membered ring.

Examples of preferred compounds of the invention include those in which:

-   -   R¹ is a phenyl or alkoxy substituted phenyl group, more        preferably a 3-alkoxy substituted phenyl group, or hydrogen;        and/or,    -   R² is hydrogen or an alkoxy group; and/or,    -   R³ is hydrogen or an alkoxy group; and/or,    -   R⁴ is ClCH₂, Cl₂CH, Cl₃C or F₃C; and/or,    -   R⁵ is hydrogen or a nitro group; and/or R⁶ is hydrogen.

Preferred compounds of the invention include compounds 31-44 as definedbelow. Especially preferred compounds are esters 39 and 40, andcompounds 43 and 44, which have the useful property of being sensitiveto visible light.

In a further aspect, the present invention provides the use of acompound as defined herein for the synthesis of a nucleic acid moleculeor peptide, wherein the compound is photolysed to produce a halogensubstituted carboxylic acid capable of removing a protecting group fromthe terminus of the nucleic acid molecule or peptide, thereby to make itreactive to chain extension by nucleosides or amino acids.

In a preferred embodiment, the free acid generated by the photolysis ofthe compounds of the invention is capable of removing a5′-O-dimethoxytrityl (DMT) protecting group present on the 5′ end of anucleic acid molecule or peptide. While the compounds can be employed inthe synthesis of nucleic acid molecules of any size starting from alinker molecule attached to a solid phase and carrying a DMT-blockedhydroxyl group at its free end or a single DMT-protected nucleotide, itis particularly useful for the synthesis of oligonucleotides in theproduction of microarrays as it allows the microarrays to be synthesisedusing readily available 5-O′-DMT protected nucleosides. This helps toameliorate the problems associated with prior art techniques whichrequire specially synthesised monomers or else generate acids which areunsuitable for the deprotection reaction, e.g. because they degrade thenucleosides within the growing oligonucleotide chain, nucleosidemonomers or undergo side reactions.

In a further aspect, the present invention provides a method ofsynthesizing a nucleic acid molecule or peptide on a solid support, themethod comprising:

-   -   (a) bringing a nucleic acid molecule or peptide having a        protected terminus into contact with a compound as defined        herein;    -   (b) photolysing the compound to produce a halogen substituted        carboxylic acid capable of removing the protecting group from        the end of the nucleic acid molecule or peptide;    -   (c) contacting the deprotected nucleic acid molecule or peptide        with nucleosides or amino acids, so that the 5′ end of the        nucleic acid molecule or peptide reacts with a nucleoside or        amino acid; and    -   (d) repeating steps (a) to (c) until the synthesis of the        nucleic acid molecule or peptide is complete.

Preferably, the compounds are used in the synthesis of a plurality ofoligonucleotides or peptides, e.g. in the formation of an array having aplurality of locations (elements) at which oligonucleotides or peptidesof a given sequence are synthesized. In this method, preferably thecompounds are used in a photodirected method of synthesising librariesof oligonucleotides or peptides, e.g. in a two-dimensional array formaton a planar glass surface.

Preferably, the compounds are immobilised in a solid polymer film toprevent or reduce acid diffusion from irradiated to non-irradiated arrayelements. Thus, in a further aspect, the present invention provides apolymeric film that comprises one or more of the above compounds. Inthis embodiment, projection photolithography defines the necessaryillumination patterns, activating the compound at defined arrayelements. The film can then be removed with solvent once irradiation andacid-dependent detritylation is completed and reaction with thenucleosides carried out.

In a further aspect, the present invention provides a method ofsynthesizing a nucleic acid or peptide array comprising a plurality ofelements on a solid support, the method employing a photoactivatableagent which is capable of photolysis to produce a deprotecting agent forremoving a protecting group from a nucleic acid molecule or peptide inthe array so that it can participate in a chain extension reaction, themethod comprising:

-   -   (a) providing the photoactivatable agent and a compound capable        of neutralising the deprotecting agent at elements in the array;    -   (b) photolysing the photoactivatable agent at the elements in        the array selected for a chain extension reaction;    -   (c) contacting the deprotected nucleic acid molecule or peptide        with nucleosides or amino acids, so that the 5′ end of the        nucleic acid molecule or peptide reacts with a nucleoside or        amino acid; and    -   (d) repeating steps (a) to (c) until the synthesis of the        nucleic acid molecule or peptide is complete.

Thus, at elements of the array selected for a chain extension reaction,an excess of the deprotecting agent is generated so that some of thedeprotecting agent is not neutralised by the neutralising agent.However, at elements of the array not selected for a chain extensionreaction, the production of deprotecting agent by stray light issubstantially neutralised by the neutralising agent.

Thus, in elements of the array selected from chain extension, an excessof the deprotecting agent will be produced as compared to thecomparatively smaller amount of the neutralising agent. However, inelements of the array which are not selected for chain extension, e.g.elements adjacent a selected element, the production of small amounts ofthe neutralising agent by stray light directed or reflected to thatelement will be largely mopped up by the neutralising agent, and willnot activate the nucleic acid molecules or peptides in those elementsfor chain extension, leading to sequence errors.

In a preferred embodiment, the photoactivatable agent is one of thephotoactivatable compounds disclosed herein which photolyse to producehalogen substituted carboxylic acid. In situations where thedeprotecting agent is an acid, the neutralising agent is conveniently aweak base or a buffer, used in an amount sufficient to neutralise theinitial production of the deprotecting compound. Conversely, where thedeprotecting agent is a base, the neutralising agent is a weak acid or abuffer.

The method described above is particularly adapted for methods ofproducing arrays in which projection lithography is used. As disclosedabove, stray light can be a problem in such methods as they generally donot employ masks.

Embodiments of the present invention will now be described in moredetail by way of example and not limitation with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the % of oligonucleotide chains without insertions,given by 100×(1−P)^(3N), plotted against contrast ratios on alogarithmic scale from 10¹ to 10⁵. Data are for N=10, 20 and 30, andexposure for 10 half-times.

FIG. 2 shows analytical reversed phase HPLC data for the products ofthree synthetic runs.

DETAILED DESCRIPTION

Effect of Stray Light in Maskless Projection Lithography In one aspect,the present invention provides a solution to a significant butpreviously unidentified problem in the art to produce high qualityarrays by a photodirected method that offers the flexibility and lowercosts of maskless projection photolithography, but without thesignificant losses of array fidelity caused by the stray lightassociated with DMA projectors or other projection systems such asliquid crystal displays and scanned laser beams modulated eitheracousto-optically or electro-optically.

This problem arises as the contrast ratio of DMD projectors is about400, some 250-fold lower than that of metal on glass masks, andnecessitates an appraisal of the effects of the higher levels of straylight in photodirected array synthesis. We have not found such anappraisal in the scientific literature, and therefore developed one, asfollows.

As already described, photodirected array synthesis (Fodor et al³⁷,Pease et al²⁹) uses photochemistry at scheduled array elements tounblock the oligonucleotide 5′-OH group of the last-coupled nucleotide,thereby allowing reaction with the 3′-phosphoramidite group of the nextadded nucleotide. Table 2 illustrates the sequence of illuminations, andshows that during the set of 4 synthetic cycles required to extend thearray from length n to n+1, each element is exposed to one period ofscheduled illumination and three periods of stray light illumination.

The photolysis of photosensitive groups used for 5′-blocking ofoligonucleotides is a unimolecular process, and for a given lightintensity the kinetics are first order. Removal of >99% of the blockinggroup requires illumination over many half-times. These half-times canvary with the oligonucleotide base by a factor of up to three³³. Theirradiation exposure is typically set at ten times the slowestbase-dependent photolytic half-time (Pease et al²⁹).

The time dependent product yield Y_(t) of a photolytic reaction withfirst order kinetics is given by the integrated rate equation (equation1; Williams & Williams⁴²) where time t is in units of photolytichalf-times:Y _(t)=1−1/exp(ln(2)t)  (2)

If the illumination period is unchanged but the intensity of light isdiminished by a factor of 1/R, where R is the contrast ratio, the valueS_(t) for the new extent of photolysis is given by:S _(t)=1−1/exp(ln(2)t/R)  (3)

These integrated rate equations (2) and (3) describe the time course inphotodirected array synthesis for the removal by scheduled and straylight respectively of a photosensitive blocking group from theoligonucleotide-5′-OH position. An example of their outcomes is given inTable 3.

As expected, Table 3 shows that the absolute rate of photolysis of aphotosensitive blocking group declines with successive half-times ofexposure, whereas if the light intensity is reduced by a large factor,the resulting much lower rate of photolysis is effectively constant overthe same period.

Calculation of the effects of stray light on the fidelity of arraysynthesis is simplified by assuming that the photolytic half-time forremoval of blocking groups is not base-specific, and that alloligonucleotide chains with unprotected 5′-OH positions are extended byone monomer on contact with nucleoside-3′-phosphoramidites.

At the start of a period of illumination all oligonucleotides are5′-blocked. The probability that a 5′-protected group will bedeprotected during a period of stray light illumination is S_(t)(equation 3). Assuming that the coupling efficiency with3′-phosphoramidites is 100%, the value of P for the probability ofsubsequent chain extension is also equal to S_(t). The number of chainsis very large, so the average number of stray-light induced additionsper chain per period of stray-light illumination, for all practicalpurposes, equals the probability P of their occurrence.

There are 3 periods of stray light illumination per array element perset of four synthetic cycles, and N such sets are required to synthesisean array of N-mers. The average number of stray-light induced additionsper chain in the completed array is therefore 3NP. Each unscheduledaddition creates a base, and increases the oligonucleotide chain lengthby one insertion. (Additions at the first and last positions of thechain are included as insertions). If there is no base-specificity forinsertions then they will be randomly distributed along the length ofthe chains.

A suitable statistical model for stray light effects on array synthesisis that of taking a set of shots at a target with a probability P pershot of hitting it. The probability distribution for 0,1,2,3 etc hitsper set of shots, or base insertions per oligonucleotide chain (creatinglengths of N, N+1, N+2, N+3 etc) is given by the binomial distribution(Bevington⁴³). The number of target molecules is very large, so thefrequency distribution of insertions is effectively equal to theprobability distribution. The most important frequency in the context ofarray synthesis is for those oligonucleotides with zero insertions andtherefore having the designed oligonucleotide sequence. Their frequencycan be obtained directly as (1−P)^(3N), where N is the chain length andP (=S_(t)) is calculated using equation (3) with defined values for thecontrast ratio and the exposure period in half-times.

FIG. 1 is a graph of the % of oligonucleotide chains without insertions,given by 100×(1−p)^(3N), plotted against contrast ratios on alogarithmic scale from 10¹ to 10⁵. Data are for N=10, 20 and 30, andexposure for 10 half-times. The highest value for contrast ratiocorresponds to the optical density of 5.0 for chrome-on-quartz masks,where the effect of stray light is negligible. The region of contrastratio from 10² to 10³ includes the values for liquid crystal displays,DMA's, and both acousto-optic and electro-optic modulators. (Modulatedand scanned laser beams could in principle be used for generation ofarray patterns). Stray light effects on array fidelity within this rangeare considerable, and increase rapidly as the contrast ratio falls.

A more specific example is given in Table 4, which presents calculatedvalues for array fidelity of a 20-mer for contrast ratios from 10² to10⁴, for exposure of 10 half-times. It illustrates the rapid degradationof array fidelity as the contrast ratio falls below 10³. At a contrastratio of 400:1, typical of the DMD projection devices⁴⁰ as used by SinghGasson et al³⁸ and LeProust et al²⁸, 35% of the chains in a 20-mer arraywould be as designed, whereas 65% would carry one or more baseinsertions. The binomial distribution in this example for chain lengthsN, N+1, N+2, N+3 and N+4 is 35, 37, 19, 6.4 and 1.6% respectively.

It is clear from these calculations (Tables 3 & 4, FIG. 1) that straylight levels associated with DMA projectors would cause significantreductions in the fidelity of oligonucleotide arrays fabricated by aprocess involving removal of photosensitive 5′-blocking groups.Photodirected oligonucleotide synthesis can also be effected by anindirect method of unblocking oligonucleotide-5′-OH groups. As describedin this application, and by others (Beecher et al³¹, Gao et al²⁷,LeProust et al²⁸) a photoacid generator is used to achieve photodirectedsynthesis. The acid, which must be confined by some means to illuminatedelements, removes oligonucleotide-5′-dimethoxytrityl groups as inconventional (non-photodirected) oligonucleotide synthesis as describedin equation (1).

Two reactions are involved in this indirect method of photodirectedsynthesis. First, the photogeneration of acid from a precursor, accordto equation (4):Photoacid generator=Acid+products  (4)

In the simplest case the reaction proceeds via an intramolecularrearrangement of a precursor, where the kinetics are first order and aredescribed by equations (2) and (3). The situation is more complex whenthe reaction mechanism is not first order, or a second acid generatingreaction is added, as with the acid catalysed thermolysis of anon-photosensitive acid precursor (Beecher et al³¹). Irrespective of themethod or mechanism, there is no obligation for photolysis and/orthermolysis of the acid generator to proceed to completion: all that isrequired is for sufficient acid to be generated for the next process,namely, the detritylation of DMT-blocked oligonucleotides according toequation (1).

Detritylation is normally effected with excess acid, and the reactionwould therefore behave kinetically as if it were first order. If thereaction does not proceed to completion during any one synthetic cycle,5′-protected oligonucleotides will remain and fail to be extended by thenext exposure to nucleoside-3′-phosphoramidite, reducing the final chainlength by 1 per failure (Temsamani et al⁴⁴, Fearon et al⁴⁵).

Exposure to acid is therefore arranged to be over many half-times of thedetritylation reaction (equation 1), say 10 or more, during which timeacid generated by stray light at unscheduled elements will cause a lowerbut virtually constant rate of detritylation. The situation withindirect photodeprotection is therefore essentially identical to thatwith the direct route. Consequently, exposure of the array to strayphotoacid for multiple detritylation half-times leads to a similaroutcome as obtains with stray-light induced 5′-deprotection overmultiple photolytic half-times.

The photoacid generated by stray light can be expected to be neutralizedby the presence of a sufficient concentration, but not more, of anappropriate buffer, or weak base, to prevent accumulation of stray lightgenerated acid to levels that cause detritylation. The amounts requiredare low, and can be calculated in the cases where equation (3) appliesby using the equation with known values for the photolytic half-time,exposure period, and contrast ratio. For example, with a contrast ratioof 400 and an exposure of 1 photolytic half-time, the amount of acidreleased by stray light would be 0.17% of the starting concentration ofphotoacid generator. The latter might typically be 100 mM, so 2-3 mMbase or buffer would be several-fold in excess of stray light inducedphotoacid, without significantly reducing the concentration of acid (50mM) arising from scheduled illumination for 1 half-time.

It should be noted that the analysis given above for the effects ofstray light on oligonucleotide 5′-deprotection is applicable tophotolytic deprotection generally when the deprotection is used as partof a synthetic strategy for fabrication of combinatorial arrays. Theanalysis for the effects of stray light on photoacid generation is alsoapplicable to photobase generation, in which case the effects can benegated by the presence of small amounts of acid or appropriate buffer.

Experimental

1. Synthesis of Photolabile Esters

1.1 Preparation of Precursors

The synthesis of the target photolabile esters required priorpreparation of various precursors shown in Scheme 1.4,5-Dimethoxy-2,6-dinitrobenzaldehyde (1) was prepared by nitration of4,5-dimethoxy-2-nitrobenzaldehyde following the literature procedure¹.The latter was made starting from 4-hydroxy-3-methoxybenzaldehyde andimproving key steps of the synthetic route described in theliterature^(2,3). Rather than following a difficult and low yieldingliterature procedure⁴, 5-chloro-2,6-dinitrobenzaldehyde (2) and5-chloro-2,4-dinitrobenzaldehyde (3) were prepared by nitration ofcommercially available 5-chloro-2-nitrobenzaldehyde. 2-Nitrobenzylalcohol (4) can be purchased from Aldrich but all the substituted benzylalcohols required as starting materials had to be prepared. Thus1-(2-Nitro-phenyl)-ethanol (5) was made by reduction of2-nitroacetophenone with sodium borohydride⁵.(2-Nitro-phenyl)-phenyl-methanol (6)^(6,7,8),(3-methoxy-phenyl)-(2-nitro-phenyl)-methanol (7),(4-methoxy-phenyl)-(2-nitro-phenyl)-methanol (8)^(9,10),(4,5-dimethoxy-2-nitro-phenyl)-(3-methoxy-phenyl)-methanol (9),(4,5-dimethoxy-2-nitro-phenyl)-phenyl-methanol (10)^(9,11),(4,5-dimethoxy-2,6-dinitro-phenyl)-phenyl-methanol (11)(5-chloro-2-nitro-phenyl)-phenyl-methanol (12)^(12,13)(5-chloro-2,6-dinitro-phenyl)-phenyl-methanol (13)(5-chloro-2,4-dinitro-phenyl)-phenyl-methanol (14) were prepared bycondensation of commercially available phenylmagnesium bromide,3-methoxyphenylmagnesium bromide or 4-methoxyphenylmagnesium bromidewith 2-nitrobenzaldehyde, 4,5-dimethoxy-2-nitrobenzaldehyde,4,5-dimethoxy-2,6-dinitrobenzaldehyde (1), 5-chloro-2-nitrobenzaldehyde,5-chloro-2,6-dinitrobenzaldehyde (2) and5-chloro-2,4-dinitrobenzaldehyde (3) in anhydrous tetrahydrofuran at−78° C.⁶. Nucleophilic displacement of the chlorine in commerciallyavailable 5-chloro-2-nitrobenzylalcohol,(5-chloro-2-nitro-phenyl)-phenyl-methanol (12)^(12,13),5-chloro-2,6-dinitro-phenyl)-phenyl-methanol (13) or(5-chloro-2,4-dinitro-phenyl)-phenyl-methanol (14), with 2Mdimethylamine in methanol, resulted in(5-dimethylamino-2-nitro-phenyl)-methanol (15)¹⁴,(5-dimethylamino-2-nitro-phenyl)-methanol (16),(5-dimethylamino-2-nitro-phenyl)-methanol (17) and(5-dimethylamino-2-nitro-phenyl)-methanol (18), respectively (Scheme 1).Initially the reaction was carried out in a small autoclave andcompounds 15 and 16 were obtained in 11-12% yield. The use of amicrowave reactor resulted in enhanced rates of the substitution andaccordingly compounds 17 and 18 were obtained in virtually quantitativeyield.

1.2. Preparation of Esters

Acetic acid and trimethylacetic acid-2-nitrobenzyl esters (20)^(15,16)and (21)¹⁷ were prepared by the reaction of appropriate acids withcommercially available 2-nitrobenzyl-chloride (19), in the presence ofsodium iodide and triethylamine¹⁵ (Scheme 1). Chloroacetic acid,dichloroacetic acid and trichloroacetic acid-2-nitrobenzyl esters(22-24)^(18,19) were obtained by esterification of commerciallyavailable 2-nitrobenzyl alcohol (4) in the presence of a catalyticamount of sulphuric acid (Scheme 2). Acetylation of compound 5 with theappropriate anhydrides, in the presence of a catalytic amount ofsulphuric acid, resulted in the corresponding α-methyl-2-nitrobenzylesters 25-29 (Scheme 2). In this series, compounds 25²⁰, 28²¹ and 29¹⁷have been reported previously. (2-Nitro-phenyl)-phenyl-methanol(6)^(6,7,8) was acetylated with acetic, chloroacetic, dichloroacetic andtrichloroacetic anhydrides (Scheme 2). The best results were obtainedwhen the acetylation was carried out in the presence of pyridine. Thecorresponding α-phenyl-2-nitrobenzyl esters 30-33 were obtained in80-90% yield after column chromatography on silicagel. Of thesynthesised compounds, in this series only α-phenyl-2-nitrobenzylacetate[acetic-acid (2-nitro-phenyl)-phenyl-methyl ester] (30) has beenreported previously⁷. The ¹³C-NMR proved particularly diagnostic in thecharacterisation of these esters, showing clearly resolved signalscorresponding to the aromatic carbons at 120-140 ppm, benzylic carbonsat 73-77 ppm and acetyl carbons between 25 and 90 ppm depending on thenumber of chlorine atoms attached. Compounds obtained in this way weremore than 95% pure by HPLC. 3-(Methoxy-phenyl)-(2-nitro-phenyl)-methanol(7) was acetylated with chloro and trichloroacetic anhydride in thepresence of pyridine. The correspondingα-(3-methoxyphenyl)-2-nitrobenzyl esters (34, 35) were obtained in80-90% yield after column chromatography on silicagel (Scheme 2).4-(Methoxy-phenyl)-(2-nitro-phenyl)-methanol (8)^(9,10) was acetylatedwith dichloro or trichloroacetic acid anhydride in the presence ofpyridine. Formation of the expected α-(4-methoxyphenyl)-2-nitrobenzylesters 36 and 37 was observed by TLC but attempts at their purificationby column chromatography on silicagel were unsuccessful and the productscould only be obtained impure. The major contaminant, however, wasisolated and its mass spectrum was consistent with4′-methoxy-2-nitrosobenzophenone (49), a product of the photolysis of 36and 37.

These results suggest that α-(4-methoxyphenyl)-2-nitrobenxylacetates areextremely photolabile and special techniques may need to be used fortheir preparation and handling.

α-Phenyl-4,5-dimethoxy-2-nitrobenzylalcohols 9, 10^(9,11) and 11 as wellas the 5-dimethylamino-2-nitrobenzylalcohols 15 , 16¹⁴, 17 and 18 wereacetylated with trichloroacetic anhydride in the presence of pyridine(Scheme 2). The new esters,α-(3-methoxyphenyl)-4,5-dimethoxy-2-nitrobenzyltrichloracetate[trichloroacetic acid(4,5-dimethoxy-2-nitro-phenyl)-(3-methoxy-phenyl)-methyl ester] (38),α-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate [trichloroaceticacid (4,5-dimethoxy-2-nitro-phenyl)-phenyl-methyl ester] (39),α-phenyl-4,5-dimethoxy-2,6-dinitrobenzyltrichloroacetate[trichloroacetic acid (4,5-dimethoxy-2,6-dinitro-phenyl)-phenyl-methylester] (40), 5-dimethylamino-2-nitrobenzyltrichloroacetate[trichloroacetic acid (5-dimethylamino-2-nitro-phenyl)-methyl ester](41) α-phenyl-5-dimethylamino-2-nitrobenzyltrichloroacetate[trichloroacetic acid (5-dimethylamino-2-nitro-phenyl)-phenyl-methylester] (42) andα-phenyl-5-dimethylamino-2,6-dinitrobenzyltrichloroacetate[trichloroacetic acid (5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methylester] (43) andα-phenyl-5-dimethylamino-2,4-dinitrobenzyltrichloroacetate[trichloroacetic acid (5-dimethylamino-2,4-dinitro-phenyl)-methyl ester](44) were isolated in excellent yields.

Their UV spectra showed absorption maxima at 345-323 nm for methoxysubstituted compounds 38, 39 and 40 and 395, 398 and 376 nm (with highextinction coefficients) for dimethylamino substituted compounds 41-44respectively. (See experimental section). All the synthesised compoundswere characterised by high resolution mass spectroscopy (HRMS),ultraviolet spectroscopy (UV) and nuclear magnetic resonancespectroscopy (NMR).

1.3. Experimental

Melting points were determined on a Reichert micro hot stage apparatusand are uncorrected. UV spectra were measured in acetonitrile with aPye-Unicam SP-8-150 UV-vis spectrophotometer. ¹H NMR spectra wererecorded at 250 MHz using a Bruker WH-250 spectrometer with TMS asinternal standard. Unless otherwise indicated, DMSO-d₆ was used as thesolvent. Mass spectra were obtained on a VG ZAB-SE spectrometer with FABionisation. Accurate masses were determined with MNOBA+Na as the matrix.HPTLC was run on Merck Kieselgel 60F₂₅₄ analytical plates in thefollowing systems: (A) CH₂Cl₂/EtOH (49:1), (B) Hexane/EtOAc (9:1), CC)Toluene. Coarse ICN silicagel was used for short column chromatography.2-Nitrobenzyl alcohol (4), 2-nitrobenzyl chloride (19)2-nitroacetophenone, phenylmagnesium bromide, 3-methoxyphenylmagnesiumbromide, 4-methoxyphenylmagnesium bromide, 2-nitrobenzaldehyde,5-chloro-2-nitrobenzylalcohol, 5-chloro-2-nitrobenzaldehyde, and4,5-dimethoxy-2-nitrobenzaldehyde were purchased from Aldrich.

1.4 Preparation of Precursors

1. Preparation of 2,6-dinitrobenzaldehyde (1) 2,6-Dinitrobenzaldehyde(1) was prepared by nitration of 3,4-dimethoxy-2-nitrobenzaldehydefollowing the literature procedure¹. 3,4-Dimethoxy-2-nitrobenzaldehydewas prepared starting from 4-hydroxy-3-methoxybenzaldehyde and modifyingkey steps such as methylation and deacetylation in the publishedliterature procedures^(2,3). The deacetylation was carried out usingconcentrated aqueous ammonia whereas dimethyl sulphate in acetone wasused for the methylation.

2. Preparation of 5-chloro-2,6-dinitrobenzaldehyde (2)5-Chloro-2-nitrobenzaldehyde (3.25 g, 17.5 mmol) was dissolved inconcentrated sulphuric acid (99%, d=1.84, 13 mL) and fuming nitric acid(d-1.501, 2.25 mL) was added dropwise over 5 min at rt. After theaddition the mixture was stirred at 55-60° C. for 6 hours. Subsequently,the mixture was cooled to rt and poured onto crushed ice (100 mL). Afterthe ice had melted, dichloromethane (150 mL) was added and the organiclayer was washed with water (2×25 mL), 3% aqueous sodium bicarbonate(4×25 mL), brine (30 mL) dried with sodium sulphate and concentrated invacuo. The residue was purified on a silicagel column eluting withhexane-ethyl acetate (3:1) to give 5-chloro-2,6-dinitrobenzaldehyde as awhite solid; yield 0.99 g, 25%, mp 101-102° C. lit ⁴102.5-103° C.;¹H-NMR δ 8.29 (d, 1H, J=8.92 Hz, H-3), 8.26 (d, 1H, J=8.92 Hz, H-3),10.2389 (s, 1H, CHO).

3. Preparation of 5-chloro-2,4-dinitrobenzaldehyde (4)5-Chloro-2-nitrobenzaldehyde (3.25 g, 17.5 mmol) was dissolved in fumingsulphuric acid (30% oleum, d=1.92, 13.5 mL) and fuming nitric acid(d=1.501, 2.5 mL) was added dropwise over 5 min at rt. After theaddition the mixture was stirred at 60-65° C. for 6 hours. Subsequently,the mixture was cooled to rt and poured onto crushed ice (100 mL). Afterthe ice had melted, dichloromethane (150 mL) was added and the organiclayer was washed with water (2×25 mL), 3% aqueous sodium bicarbonate(4×25 mL), brine (30 mL) dried (sodium sulphate) and concentrated invacuo. The residue was purified on a silicagel column eluting withhexane-ethyl acetate (17:3) to give 5-chloro-2,4-dinitrobenzaldehyde asa white solid, mp indef; yield 0.82 g, (21%);); observed FAB MS230.9818, [C₇H₄ ClN₂O₅+H]⁺ requires 230.9809; ¹H-NMR δ 8.22 (s, 1H,H-3), 8.93 (s, 1H, H-4), 10.2647 (s, 1H, CHO)

4. Preparation of (2-nitro-phenyl)-phenyl-methanol (6)(3-methoxy-phenyl)-(2-nitrophenyl)-methanol (7),(4-methoxy-phenyl)-(2-nitro-phenyl)-methanol (8)(4,5-dimethoxy-2-nitro-phenyl)-(3-methoxy-phenyl)-methanol (9),(4,5-dimethoxy-2-nitro-phenyl)-phenyl-methanol (10)(4,5-dimethoxy-2,6-dinitro-phenyl)-phenyl-methanol (11) and(5-chloro-2-nitro-phenyl)-phenyl-methanol (12)(5-chloro-2,6-dinitro-phenyl)-phenyl-methanol (13)(5-chloro-2,4-dinitro-phenyl)-phenyl-methanol (14) (General Procedure)

2-Nitrobenzaldehyde, 4,5-dimethoxy-2-nitro-benzaldehyde,4,5-dimethoxy-2,6-dinitro-benzaldehyde (1),5-chloro-2-nitrobenzaldehyde, 5-chloro-2,6-dinitrobenzaldehyde (2), or5-chloro-2,4-dinitrobenzaldehyde (3), (10 mmol) was dissolved inanhydrous tetrahydrofuran (50 mL) and the solution was cooled to −78° C.Phenylmagnesium bromide, 3-methoxyphenylmagnesium bromide or4-methoxyphenyl magnesium bromide (1M solution in THF, 10 mL) was addedto the stirred solution by syringe during 15 minutes. After 10 minutesat −78° C. the mixture was stirred at −15° C. for 15 minutes. 2% Aqueoushydrochloric acid (100 mL) was added dropwise over 20 minutes followedby dichloromethane (100 mL). Each solution was washed with water (50mL), 3% aqueous sodium bicarbonate (4×50 mL), water (50 mL), brine (50mL), dried with anhydrous sodium sulphate and concentrated in vacuo.Each residue was chromatographed on silicagel eluting withdichloromethane/ethanol or dichloromethane to give compounds 6-14.

(2-Nitro-phenyl)-phenyl-methanol (6): yield 81%; Rf (A) 0.42; yellowoil; ¹H-NMR δ 6.19 (bs, 2H, CH, OH), 7.26 (m, 5H, H-2′-H-6′), 7.42-7.90(m, 4H, H-3-H-6).

(3-Methoxy-phenyl)-(2-nitrophenyl)-methanol (7): yield 83%; Rf (A) 0.36;yellow oil; ¹H-NMR δ 3.71 (s, 3H, OCH₃), 6.22 (m, 2H, CH, OH), 6.84 (m,3H, H-2′, H-4′, H-6′), 7.22 (m, 1H, H-5′), 7.51 (m, 1H, H-4), 7.74 (m,2H, H-5, H-6), 7.92 (m, 1H, H-3); UV λ_(max) 257 nm ε 4993, λ_(min) 238nm.

(4-Methoxy-phenyl)-(2-nitro-phenyl)-methanol (8): yield 55%; Rf (A)0.31; colourless oil; observed FAB MS 294.0755, [C₁₅H₁₃NO4+Na]⁺ requires294.0742; ¹H-NMR δ 3.71 (s, 3H, OCH₃), 6.09 (d, 1H, CH, J=4.68 Hz), 6.16(d, 1H, OH, J=4.68 Hz), 6.85 (m, 2H, H-3′, H-5′), 7.14 (m, 2H, H-2′,H-6′), 7.51 (m, 1H, H-4), 8.01(m, 3H, H-3, H-5, H-6).

(4,5-Dimethoxy-2-nitro-phenyl)-(3-methoxy-phenyl)-methanol (9): yield81%; Rf (A) 0.25; reddish oil; δ 3.66 (s, 3H, OCH₃), (3.84 (s, 3H, OCH₃)3.87 (s, 3H, OCH₃), 6.18 (d, 1H, OH, J=5.46 Hz), 6.29 (m, 2H, CH, H-2′),6.80 (m, 3H, H-4′-H-6′), 7.40 (s, 1H, H-6), 7.55 (s, 1H, H-3).

(4,5-Dimethoxy-2-nitro-phenyl)-phenyl-methanol (10): yield 69%; Rf (A)0.34; reddish oil; observed FAB MS 312.0846, [C₁₅H₁₅NO₅+Na]⁺ requires312.0848; ¹H-NMR δ 3.84 (s, 3H, OCH₃) 3.88 (s, 3H, OCH₃), 6.16 (d, 1H,OH, J=5.03 Hz), 6.30 (d, 1H, CH, J=5.03 Hz) 7.23 (m, 5H, H-2′-H-6′),7.45 (s, 1H, H-6), 7.56 (s, 1H, H-3); UV λ_(max) 257 nm ε 4993, λ_(min)238 nm.

(4,5-dimethoxy-2,6-dinitro-phenyl)-phenyl-methanol (11): yield 56%; Rf(A) 0.27; light orange solid; mp indef; observed FAB MS 357.0710,[C₁₅H₁₅N₂O₇+H]⁺ requires 357.0699; ¹H-NMR δ 3.90 (s, 3H, OCH₃) 3.99 (s,3H, OCH₃), 6.01 (d, 1H, CH, J=3.08 Hz), 6.72 (d, 1H, CH, J=3.08 Hz),7.23 (m, 5H, H-2′-H-6′), 7.86 (s, 1H, H-3).

(5-Chloro-2-nitro-phenyl)-phenyl-methanol (12): yield 94%; Rf (A) 0.29;light tan oil; ¹H-NMR δ 6.36 (d, 1H, OH, J=8.01 Hz), 6.73 (d, 1H, CH,J=8.01 Hz), 7.28 (m, 5H, H-2′-H-6′), 7.64 (m, 1H, H-6), 7.82 (m, 1H,H-4) 7.94 (m, 1H, H-3).

(5-chloro-2,6-dinitro-phenyl)-phenyl-methanol (13): yield 83%; Rf (A)0.32; colourless solid; ¹H-NMR δ 6.11 (d, 1H, CH, J=4.91 Hz), 6.84 (d,1H, OH, J=4.91 Hz), 7.30 (m, 5H, H-2′-H-6′), 8.06 (d, 1H, J=8.81 Hz,H-4), 8.20 (d, 1H, J=8.81 Hz, H-3).

(5-chloro-2,4-dinitro-phenyl)-phenyl-methanol (14): yield 48%; Rf (A)0.40; light yellow oil; ¹H-NMR δ 6.26 (d, 1H, CH, J=4.80 Hz), 6.60 (d,1H, OH, J=4.80 Hz), 7.31 (m, 5H, H-2′-H-6′), 8.17 (s, 1H, H-6), 8.73 (s,1H, H-3).

5. Preparation of (5-dimethylamino-2-nitro-phenyl)-methanol (15) and(5-dimethylamino-2-nitro-phenyl)-phenyl-methanol (16)

(5-Chloro-2-nitro-phenyl)-methanol (0.5 g, 2.67 mmol) or(5-chloro-2-nitro-phenyl)-phenyl-methanol (12) (0.5 g, 1.90 mmol) weretreated with 2M solution of dimethylarnine in methanol (6 mL) and thesolution was heated in a small autoclave at 60-65° C. for 18 hours. Thesolvent was removed in vacuo and each residue was coevaporated withdichloromethane (3×10 mL) and applied onto a column of silicagel. Thecolumn was eluted with dichloromethane/ethanol (49:1) to give compounds15 and 16.

(5-Dimethylamino-2-nitro-phenyl)-methanol (15): yield 11%; Rf (A) 0.30;yellow oil; ¹H-NMR δ 3.08 (s, 6H, N (CH₃)₂, 4.84 (d, 2H, J=4.85 Hz,CH₂), 5.42 (t, 1H, J=4.85 Hz, OH), 6.67 (m, 1H, H-6), 7.04 (m, 1H, H-4),8.04 (d, 1H, J=9.12 Hz, H-3); UV λ_(max) 395 nm ε 17163, λ_(min) 292 nm.

(5-Dimethylamino-2-nitro-phenyl)-phenyl-methanol (16): yield 12%; Rf (A)0.12; yellow solid, mp 95-97° C.; observed FAB MS 272.1173,[C₁₅H₁₆N₂O₃+H]⁺ requires 272.1161; ¹H-NMR δ 3.09 (s, 6H, N(CH₃)₂), 6.00(d, 1H, J=5.19 Hz, OH), 6.47 (d, 1H, J=5.19 Hz, CH), 6.70 (m, 1H, H-6)7.21 (m, 6H, H-2′-H-6′, H-4), 7.98 (d, 1H, J=9.32 Hz, H-3); UV λ_(max)398 nm, ε 11435, λ_(min) 322 nm.

6. Preparation of (5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methanol(17) and (5-dimethylamino-2,4-dinitro-phenyl)-methanol (18)

(5-Chloro-2,6-dinitro-phenyl)-phenyl-methanol (13) (0.5 g, 1.90 mmol) or(5-chloro-2,4-dinitro-phenyl)-phenyl-methanol (14) (0.5 g, 2.67 mmol)were treated with 2M solution of dimethylamine in methanol (6 mL) andthe solution was heated in a microwave reactor (CEM Focused Microwave™Synthesis System Model Discover) at 50-55° C. for 15 minutes. Thesolvent was removed in vacuo and each residue was coevaporated withdichloromethane (2×10 mL) and applied onto a column of silicagel. Thecolumn was eluted with dichloromethane/ethanol (49:1) to give compounds17 and 18.

(5-Dimethylamino-2,6-dinitro-phenyl)-phenyl-methanol (17): yield 94%; Rf(A) 0.22; yellow solid, mp indef; observed FAB MS 340.0895[C₁₅H₁₅N₃O₅+Na]⁺ requires 340.0909; ¹H-NMR δ 2.85 (s, 6H, N(CH₃)₂), 5.99(d, 1H, J=5.07 Hz, CH), 6.49 (d, 1H, J=5.07 Hz, OH), 7.24 (m, 6H,H-2′-H-6′, H-4), 7.99 (d, 1H, J=9.31 Hz, H-3); UV λ_(max) 387 nm ε11150.

(5-Dimethylamino-2,4-dinitro-phenyl)-phenyl-methanol (18): yield 91%; Rf(A) 0.35; reddish solid, mp 147-150° C.; observed FAB MS 318.1104,[C₁₅H₁₅N₃O₅+H]⁺ requires 318.1090; ¹H-NMR δ 3.04 (s, 6H, N (CH₃)₂, 6.30(d, 1H, J=5.17 Hz, CH), 6.44 (d, 1H, J=5.17 Hz, OH), 7.27 (m, 5H,H-2′-H-6′), 7.64 (s, 1H, H-4); 8.52 (s, 1H, H-3); ¹³C-NMR δ 42.53[N(CH₃)₂], 70.61 (CH), 116.22 (C-6), 126.32 (C-3), 127.86 (C-2′, C-4′,C-6′), 128.54 (C-3′, C-5), 142.84 (C-4), 145.73 (C-2), 148.14 (C-5); UVλ_(max) 377 nm ε 16080.

1.5. Preparation of Esters

1. Synthesis of acetic acid 2-nitro-benzyl ester (20) andtrimethyl-acetic acid 2-nitro-benzyl ester (21) (General Procedure)

2-Nitrobenzylchloride (19) (1.72 g, 10 mmol) and sodium iodide (100 mg)were suspended in dry ethyl acetate (50 mL). Triethylamine (2.02 g, 2.5mL) and glacial acetic acid (1.6 g, 2 mL , 20 mmol ) or pivaloic acid(2.04 g, 20 mmol) were added to the suspension and the mixture washeated under reflux for 20 h. The solid was filtered off (glassmicrofibre filter), the filtrate was washed with 1M hydrochloric acid(20 mL), water (2×20 mL), 3% aqueous sodium bicarbonate (2×20 mL), water(20 mL) and brine (20 mL), dried with sodium sulphate and concentratedin vacuo. Each residue was purified by column chromatography onsilicagel eluting with dichloromethane/ethanol (500:1) to give products20 and 21.

Acetic acid 2-nitro-benzyl ester (20): yield 1.58 g (81%); Rf (C) 0.22;yellow solid, mp 53-55° C.; observed FAB MS 196.0606, [C₉H₉NO₄+H]⁺requires 196.0610; ¹H-NMR δ 2.11 (s, 3H, CH₃CO), 5.40 (s, 2H, CH₂),7.59-7.82 (m, 3H, H-4, H-5, H-6), 8.09-8.13 (m, 1H, H-3); UV λ_(max) 259nm ε 4864, λ_(min) 232 nm.

Trimethyl-acetic acid 2-nitro-benzyl ester (21): yield 1.72 g (82%); Rf(C) 0.40; yellow oil; observed FAB MS 238.1085 [C₁₂H₁₆NO₄+H]⁺ requires238.1079; ¹H-NMR (CDCl₃) δ 1.16 [s, 9H, (CH₃)₃C], 5.39 (s, 2H, CH₂),7.59-7.85 (m, 3H, H-4, H-5, H-6), 8.07-8.11 (m, 1H, H-3); UV λ_(max) 259nm ε 4776, λ_(min) 235 nm.

2. Synthesis of chloro-acetic acid 2-nitro-benzyl ester (22),dichloro-acetic acid 2-nitro-benzyl ester (23) and trichloro-acetic acid2-nitro-benzyl ester (24) (General Procedure)

2-Nitrobenzylalcohol (1.53 g, 10 mmol) and chloroacetic acid (30 mmol),dichloroacetic acid (60 mmol) or trichloroacetic acid (50 mmol) weredissolved in toluene (50 mL) and concentrated sulphuric acid (0.5 mL)was added. Each mixture was heated for 4 hours at 80-90° C. andsubsequently chloroform (150 mL) was added. Each solution was washedwith water (50 mL), 3% aqueous sodium bicarbonate (4×50 mL), water (50mL), brine (50 mL), dried with sodium sulphate and concentrated invacuo. Each residue was chromatographed on silicagel eluting withdichloromethane to give compounds 22-24.

Chloro-acetic acid 2-nitro-benzyl ester (22): yield 72%; Rf (A) 0.86;yellow oil; observed FAB MS 230.0213, [C₉H₈ClNO₄+H]⁺ requires 230.0220;¹H-NMR δ 4.53 (s, 2H, ClCH₂CO), 5.54 (s, 2H, CH₂), 7.61-7.84 (m, 3H,H-4, H-5, H-6), 8.12-8.16 (m, 1H, H-3); UV λ_(max) 259 nm ε 5120,λ_(min 234) nm.

Dichloro-acetic acid 2-nitro-benzyl ester (23): yield 78%; Rf (A) 0.88;yellow oil; observed FAB MS 263.9821.0213, [C₉H₇Cl₂NO4+H]⁺ requires263.983; ¹H-NMR (CDCl₃) δ 5.72 (s, 2H, CH₂), 6.06 (s, 1H, Cl₂CHCO),7.54-7.72 (m, 3H, H-4, H-5, H-6), 8.16-8.19 (m, 1H, H-3); UV λ_(max) 259nm ε 4159, λ_(min) 235 nm.

Trichloro-acetic acid 2-nitro-benzyl ester (24); yield 70%; Rf (A) 0.89;yellow oil; observed FAB MS 297.9440 [C₉H₆Cl₃NO₄+H]⁺ requires 297.9445;¹H-NMR (CDCl₃) δ, 5.79 (s, 2H, CH₂), 7.67-7.89 (m, 3H, H-4, H-5, H-6),8.17-8.20 (m, 1H, H-3); UV λ_(max) 259 nm ε 4975, λ_(min) 232 nm.

3. Synthesis of acetic acid 1-(2-nitro-phenyl)-ethyl ester (25),chloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (26), dichloro-aceticacid 1-(2-nitro-phenyl)-ethyl ester (27), trichloro-acetic acid1-(2-nitro-phenyl)-ethyl ester (28) and trimethyl-acetic acid1-(2-nitro-phenyl)-ethyl ester (29) (General Procedure)

1-(2-Nitro-phenyl)-ethanol (5) (1.7 g, 10 mmol) was added dropwise over10 minutes to a stirred solution of acetic anhydride, chloroaceticanhydride, dichloroacetic anhydride, trichloroacetic anhydride ortrimethylacetic anhydride (50 mmol) with the addition of concentratedsulphuric acid (0.5 mL) in toluene (25 mL) at 0° C. After 1 hour at roomtemperature, each mixture was heated at 80-90° C. for 3 hours andsubsequently ethyl acetate (100 mL) was added. Each solution was washedwith water (50 mL), 3% aqueous sodium bicarbonate (4×50 mL), water (50mL) brine (50 mL), dried with sodium sulphate, and concentrated invacuo. Each residue was chromatographed on silicagel eluting withdichloromethane to give compounds 25-29.

Acetic acid 1-(2-nitro-phenyl)-ethyl ester (25): yield 61%; Rf (A) 0.81;yellow oil; observed FAB MS 210.0775, [C₁₀H₁₁NO₄+H]⁺ requires 210.0766;¹H-NMR δ 1.56 (d, 3H, J=6.90 Hz, CH₃), 2.01 (s, 3H, CH₃CO), 6.07 (q, 1H,J=6.90 Hz, CH), 7.53-7.59 (m, 1H, H-4), 7.70-7.77 (m, 2H, H-5, H-6),7.94-7.97 (m, 1H, H-3); UV λ_(max) 256 nm ε 4569 λ_(min) 235 nm.

Chloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (26): yield 34%; Rf(A) 0.73; yellow oil; observed FAB MS 266.0201, [C₁₀H₁₀ClNO₄+Na]⁺requires 266.0196; ¹H-NMR δ 1.61 (d, 3H, J=6.39 Hz, CH₃), 4.43 (s, 1H,ClCH₂CO), 6.18 (q, 1H, J=6.39 Hz, CH), 7.62-7.88 (m, 3H, H-4, H-5, H-6),8.02-8.09 (m, 1H, H-3); UV λ_(max) 256 nm ε 5067, λ_(min) 234 nm.

Dichloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (27): yield 64%; Rf(A) 0.83; yellow oil; observed FAB MS 277.9980, [C₁₀H9Cl₂NO₄+H]⁺requires 277.9987; ¹H-NMR δ 1.67 (d, 3H, J=7.45 Hz, CH₃), 6.26 (q, 1H,J=7.45 Hz, CH), 6.93 (s, 1H, Cl₂CHCO, 7.62-7.88 (m, 3H, H-4, H-5, H-6),8.03-8.07 (m, 1H, H-3); UV λ_(max) 256 nm ε 4536, λ_(min) 235 nm.

Trichloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (28): yield 64%; Rf(A) 0.91; colourless oil; observed FAB MS 333.9407, [C₁₀H8Cl₃NO₄+Na]⁺requires 333.941; ¹H-NMR δ 1.73 (d, 3H, J=6.48 Hz, CH₃), 6.37 (q, 1H,J=6.48 Hz, CH), 7.62-7.88 (m, 3H, H-4, H-5, H-6), 8.02-8.09 (m, 1H,H-3); UV λ_(max) 256 nm ε 4520, λ_(min) 234 nm.

Trimethyl-acetic acid 1-(2-nitro-phenyl)-ethyl ester (29): yield 64%; Rf(A) 0.91; colourless oil; observed FAB MS 252.1226 [C₁₃H₁₈NO₄+H]⁺requires 252.1236; ¹H-NMR δ 1.19 [s, 9H, C (CH₃)₃], 1.73 (d, 3H, J=6.52Hz, CH₃), 6.07 (q, 1H, J=6.52 Hz, CH), 7.55-7.59 (m, 1H, H-4), 7.67-7.77(m, 2H, H-5, H-6), 7.93-7.97 (m, 1H, H-3); UV λ_(max) 251 nm ε 6250,λ_(min) 237 nm.

4. Synthesis of acetic acid (2-nitro-phenyl)-phenyl-methyl ester (30),chloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (31),dichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (32) andtrichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (33) (GeneralProcedure)

(2-Nitro-phenyl)-phenyl-methanol (6) (2.29 g, 10 mmol) and aceticanhydride, chloroacetic anhydride, dichloroacetic anhydride ortrichloroacetic anhydride (50 mmol) were dissolved in drydichloromethane (20 mL) and pyridine (0.5 mL) was added by syringe underargon. Each mixture was stirred at room temperature for 8 hours andsubsequently ethyl acetate (100 mL) was added. Each solution was washedwith water (50 mL), 3% aqueous sodium bicarbonate (4×50 mL), water (50mL), brine (50 mL), dried with sodium sulphate, and concentrated invacuo. Each residue was chromatographed on silicagel eluting withdichloromethane to give compounds 30-33.

Acetic acid (2-nitro-phenyl)-phenyl-methyl ester (30): yield 83%; Rf (A)0.91; yellow oil; observed FAB MS 294.0755, [C₁₅H₁₃NO₄+Na]⁺ requires294.0742; ¹H-NMR δ 2.11 (s, 3H, CH₃CO), 7.24 (s, 1H, CH), 7.31-7.40 (m,5H, H-2′-H-6′), 7.61-7.79 (m, 3H, H-4, H-5, H-6), 8.00-8.03 (m, 1H,H-3); ¹³C-NMR δ 20.98 (CH₃), 72.01 (CH), 125.02 (C-3), 127.80 (C-4′),128.78 (C-2′, C-6′), 128.95 (C-3′, C-5′), 128.98 (C-4), 129.80 (C-6),134.30 (C-5), 134.56 (C-1), 138.43 (C-1′), 148.29 (C-2), 169.89 (CO); UVλ_(max) 257 nm ε 4993, λ_(min) 238 nm.

Chloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (31): yield 90%;Rf (B) 0.17; yellow oil; ¹H-NMR δ 4.53 (s, 2H, ClCH₂CO), 7.31-7.44 (m,6H, H-2′-H-6′, CH), 7.61-7.85 (m, 3H, H-4, H-5, H-6), 8.04-8.08 (m, 1H,H-3); ¹³C-NMR δ 65.17 (CHCl₂), 75.14 (CH), 125.22 (C-3), 127.89 (C-4′),128.91 (C-2′, C-6′), 129.00 (C-3′, C-5′), 129.03 (C-4), 130.05 (C-6),133.95 (C-5), 134.51 (C-1), 137.81 (C-1′), 148.10 (C-2), 166.93 (CO); UVλ_(max) 257 nm ε 4498, λ_(min) 238 nm.

Dichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (32): yield63%; Rf (B) 0.19; yellow oil; ¹H-NMR δ 7.03 (s, 1H, Cl₂CHCO), 7.36-7.41(m, 6H, CH, H-2′-H-6′), 7.64-7.70 (m, 2H, H-4, H-6), 7.81-7.85 (m, 1H,H-5), 8.08-8.12 (m, 1H, H-3); ¹³C-NMR δ55.26 (CH₂Cl), 73.57 (CH), 125.46(C-3), 127.87 (C-4′), 128.85 (C-2′, C-6′), 129.14 (C-3′, C-5′),129.27(C-4), 130.41 (C-6), 133.16 (C-5), 134.55 (C-1), 137.09 (C-1′),148.10 (C-2), 163.65 (CO); UV λ_(max) 257 nm ε 4156, λ_(min) 241 nm.

Trichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (33): yield69%; Rf (B) 0.21; yellow oil; ¹H-NMR δ 7.44 (bs, 5H, arom), 7.51 (s, 1H,CH) 7.62-7.70 (m, 2H, H-4, H-6,), 7.86 (m, 1H, H-5), 8.12 (m, 1H, H-3);¹³C-NMR δ 77.46 (CH), 89.49 (CCl₃), 125.63 (C-3), 127.88 (C-4′), 128.96(C-2′, C-6′), 129.23 (C-3′, C-5′), 129.50 (C-4), 130.69 (C-6), 132.59(C-5), 134.76 (C-1), 136.53 (C-1′), 148.18 (C-2), 160.33 (CO); UVλ_(max) 257 nm ε 4442, λ_(min) 238 nm.

5. Synthesis of chloro-acetic acid(3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(34) andtrichloro-acetic acid (3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(35) (General Procedure)

(3-Methoxy-phenyl)-(2-nitro-phenyl)-methanol (7) (2.59 g, 10 mmol) andchloroacetic anhydride or trichloroacetic anhydride (50 mmol) weredissolved in dry dichloromethane (20 mL) and pyridine (0.5 mL) was addedby syringe under argon. Each mixture was stirred at room temperature for8 hours and subsequently ethyl acetate (100 mL) was added. Each solutionwas washed with water (50 mL), 3% aqueous sodium bicarbonate (4×50 mL),water (50 mL), brine (50 mL), dried with sodium sulphate andconcentrated in vacuo. Each residue was chromatographed on silicageleluting with dichloromethane to give compounds 34 and 35.

Chloro-acetic acid (3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(34): yield 91%; yellow oil; Rf (A), 0.67; observed FAB MS 335.0573,[C₁₆H₁₄ClNO₅+Na]⁺ requires 335.0561; ¹H-NMR δ 3.76 (s, 3H, OCH₃), 4.27(s, 1H, ClCH₂CO), 6.91-6.97 (m, 3H, CH, H-2′-H-4′), 7.30-7.35 (m, 2H,H-5′, H-6′), 7.66-7.82 (m, 2H, H-4, H-6), 7.85-7.91 (m, 1H, H-5),8.05-8.09 (m, 1H, H-3); UV λ_(max) 266 nm ε 4792, λ_(min) 243 nm.

Trichloro-acetic acid (3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(35): yield 93%; yellow oil; Rf (A) 0.75; observed FAB MS 404.62[C₁₆H₁₂Cl₃NO₅+Na]⁺ requires 404.63; ¹H-NMR δ 3.73 (s, 3H, OCH₃),6.97-7.09 (m, 3H, CH, H-2′-H-4′), 7.36 (m, 1H, H-6′), 7.53-7.57 (m, IH,H-5′), 7.69-7.73 (m, 2H, H-4, H-6), 7.80-7.84 (m, 1H,H-5), 8.11-8.15 (m,1H, H-3); UV λ_(max) 265 nm ε 5091, λ_(min) 244 nm.

6. Synthesis of dichloro-acetic acid(4-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester (36) andtrichloro-acetic acid (4-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(37) (General Procedure)

(4-Methoxy-phenyl)-(2-nitro-phenyl)-methanol (8) (2.59 g, 10 mmol) anddichloroacetic anhydride or trichloroacetic anhydride (50 mmol) weredissolved in dry dichloromethane (20 mL) and pyridine (0.5 mL) was addedby syringe under argon. Each mixture was stirred at room temperature for8 hours and subsequently ethyl acetate (100 mL) was added. Each solutionwas washed with water (50 mL), 3% aqueous sodium bicarbonate (4×50 mL),water (50 mL), brine (50 mL), dried with sodium sulphate andconcentrated in vacuo. Each residue was chromatographed on silicageleluting with dichloromethane to give compounds 36 and 37.

Dichloro-acetic acid (4-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(36) (obtained impure due to instability): yield 81%; yellow oil rapidlydecomposing on storage; Rf (A) 0.71; ¹H-NMR δ 3.77 (s, 3H, OCH₃)6.96-7.05 (m, 2H, H-3′, H-5′), 7.09 (s, 1H, Cl₂CHCO), 7.36-7.41 (m, 3H,CH, H-2′, H-6′), 7.66-7.73 (m, 2H, H-4, H-6), 7.85-7.91 (m, 1H, H-5),8.09-8.14 (m, 1H, H-3).

Trichloro-acetic acid (4-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(37) (obtained impure due to instability): yield 93%; yellow oil rapidlydecomposing on storage; Rf (A) 0.78; ¹H-NMR δ 3.75 (s, 3H, OCH₃)6.96-7.11 (m, 2H, H-3′, H-5′), 7.28-7.34 (m, 2H, H-2′, H-6′), 7.44 (s,1H, CH), 7.64-8.30 (m, 4H, H-3-H-6); UV λ_(max) 267 nm ε 11504 nm,λ_(min) 246 nm.

7. Synthesis of trichloro-acetic acid(4,5-dimethoxy-2-nitrophenyl)-(3-methoxyphenyl)-methyl ester (38),trichloro-acetic acid (4,5-dimethoxy-2-nitrophenyl)-phenyl-methyl ester(39)) and trichloro-acetic acid (4,5-dimethoxy-2,6-dinitrophenyl)-phenyl-methyl ester (40) (GeneralProcedure)

(4,5-Dimethoxy-2-nitrophenyl)-(3-methoxyphenyl)-methanol (7) (3.19 g, 10mmol) or (4,5-dimethoxy-2-nitrophenyl)-phenyl-methanol (10) (2.89 g, 10mmol) or (4,5-dimethoxy-2,6-dinitrophenyl)-phenyl-methanol (11) (2.89 g,10 mmol) and trichloroacetic anhydride (50 mmol) were dissolved in drydichloromethane (20 mL) and pyridine (0.5 mL) was added by syringe underargon. Each mixture was stirred at room temperature for 6 hours andsubsequently ethyl acetate (100 mL) was added. Each solution was washedwith water (50 mL), 3% aqueous sodium bicarbonate (4×50 mL), water (50mL), brine (50 mL), dried with sodium sulphate and concentrated invacuo. Each residue was chromatographed on silicagel eluting withdichloromethane to give compounds 38-40.

Trichloro-acetic acid(4,5-dimethoxy-2-nitrophenyl)-(3-methoxyphenyl)-methyl ester (38); yield81%; yellow oil; Rf (A) 0.87; ¹H-NMR δ 3.73 (s, 3H, OCH₃), 3.82 (s, 3H,OCH₃) 3.89 (s, 3H, OCH₃) 6.92-7.03 (m, 4H, CH, H-2′, H-4′, H-6′),7.30-7.37(m, 1H, H-5′), 7.52 (s, IH, H-6), 7.73 (s, 1H, H-3); UV λ_(max)344 nm ε 5064, λ_(min) 246 nm.

Trichloro-acetic acid (4,5-dimethoxy-2-nitrophenyl)-phenyl-methyl ester(39); yield 67%; yellow solid; Rf (A) 0.74; observed FAB MS 432.9871,[C₁₇H₁₄Cl₃NO₆]⁺ requires 432.9887; ¹H-NMR δ 3.84 (s, 3H, OCH₃), 3.89 (s,3H, OCH₃), 7.09 (s, H, CH), 7.42 (m, 5H, H-2′-H-6′), 7.55 (s, 1H, m,H-6), 7.74 (s, 1H, H-3); UV λ_(max) 345 nm ε 5250, λ_(min) 272 nm.

Trichloro-acetic acid (4,5-dimethoxy-2,6-dinitrophenyl)-phenyl-methylester (40); yield 79%; yellow solid, mp 105-106° C.; Rf (A) 0.79;observed FAB MS 611.8800, [C₁₇H₁₃Cl₃N₂O₈+Cs]⁺ requires 611.8792; ¹H-NMRδ 3.92 (s, 3H, OCH₃), 4.04 (s, 3H, OCH₃), 7.10 (s, H, CH), 7.28-7.42 (m,6H, H-2′-H-6′, CH), 8.03 (s, 1H, H-5); ¹³C-NMR δ 57.88 (OCH₃), 62.79(OCH₃), 74.88 (CH), 89.16 (CCl₃), 112.11 (C-4), 115.01(C-1), 126.56(C-3′, C-5′), 128.77 (C-2′, C-6′), 129.07 (C-4′), 135.40 (C-1′), 144.27(C-2), 144.73 (C-6), 145.36 (C-4), 153.89 (C-5), 160.95 (CO); UV λ_(max)323 nm ε 4110.

8. Synthesis of trichloro-acetic acid 5-dimethylamino-2-nitro-benzylester (41), trichloro-acetic acid(5-dimethylamino-2-nitro-phenyl)-phenyl-methyl ester (42),trichloro-acetic acid (5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methylester (43) and trichloro-acetic acid(5-dimethylamino-2,4-dinitro-phenyl)-phenyl-methyl ester (44) (GeneralProcedure)

(5-Dimethylamino-2-nitro-phenyl)-methanol (15) (0.217 g, 1 mmol),(5-dimethylamino-2-nitro-phenyl)-phenyl-methanol (16) (0.272 g, 1 mmol),(5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methanol (17) (0.317 g, 1mmol) or (5-dimethylamino-2-nitro-phenyl)-phenyl-methanol (18) (0.317 g,1 mmol) and trichloroacetic anhydride (5 mmol) were dissolved in drydichloromethane (5 mL) and pyridine (0.1 mL) was added by syringe underargon. Each mixture was stirred at room temperature for 6 hours andsubsequently ethyl acetate (50 mL) was added. Each solution was washedwith water (5 mL), 3% aqueous sodium bicarbonate (4×5 mL), water (5 mL),brine (5 mL), dried with sodium sulphate and concentrated in vacuo. Eachresidue was chromatographed on silicagel eluting with dichloromethane togive compounds 41-44.

Trichloro-acetic acid (5-dimethylamino-2-nitro-phenyl)-phenyl-methylester (41): yield 78%; yellow froth; Rf (A) 0.85; ¹H-NMR δ 3.08 [s, 6H,N(CH₃)₂], 6.84 (m, 2H, H-4, H-6) 7.39 (m, 5H, H-2′-H-6′), 7.72 (s, 1H,CH), 8.13 (d, 1H, J=9.40 Hz, H-3); UV λ_(max) 398 nm ε 18344, λ_(min)295 nm.

Trichloro-acetic acid 5-dimethylamino-2-nitro-benzyl ester (42): yield44%; yellow foam; Rf (A), 0.88; observed FAB MS 472.8842,[C₁₁H₁₁Cl₃N₂O₄Cs]⁺ requires 472.8839; ¹H-NMR δ 3.08 [s, 6H, N(CH₃)₂],5.78 (s, 2H, CH₂), 6.82 (m, 2H, H-4, H-6), 8.04 (d, 1H, J=9.20 Hz, H-3);UV λ_(max) 395 nm ε 16680, λ_(min) 292 nm.

Trichloro-acetic acid (5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methylester (43): yield 80%; yellow solid, mp 129-130° C.; Rf (A) 0.85;observed FAB MS 462.0040, [C17H₁₄Cl₃N₃O₆+H]⁺ requires 462.0026; ¹H-NMR δ3.08 [s, 6H, N(CH₃)₂], 6.84 (m, 2H, H-4, H-6) 7.39 (m, 5H, H-2′-H-6′),7.72 (s, 1H, CH), 8.13 (d, 1H, J=9.40 Hz, H-3); ¹³C-NMR δ 41.72[N(CH₃)₂], 76.05 (CH), 89.24 (CCl₃), 119.14 (C-4), 126.59 (C-3′, C-5′),128.54 (C-2′, C-3, C-6′), 128.84 (C-4′), 136.64 (C-1′), 137.68 (C-1),138.33 (C-2), 148.30 (C-5), 161.27 (CO); UV λ_(max) 376 nm ε 11000.

Trichloro-acetic acid (5-dimethylamino-2,4-dinitro-phenyl)-phenyl-methylester (44): yield 73%; yellow solid, mp 91-92° C.; Rf (A) 0.68; observedFAB MS 593.8980, [C17H₁₄Cl₃N₃O₆+Cs]⁺ requires 593.9003; ¹H-NMR δ 3.01[s, 6H, N(CH₃)₂], 7.23 (s, 1H,CH); 7.42 (m, 5H, H-2′-H-6′), 8.67 (s, 1H,H-3); ¹³C-NMR δ 42.61 [N(CH₃)₂], 77.99 (CH), 89.51 (CCl₃), 115.57 (C-6),127.21(C-3), 128.73 (C-3′, C-5′), 129.17 (C-2′, C-6′), 129.74 (C-4′),134.06 (C-1), 134.43 C-1′), 135.71 (C-2), 138.17 (C-4), 148.06 (C-5),160.15 (CO); UV λ_(max) 376 nm ε 16000.

2. Irradiation of Photolabile Esters

2.1 Results

Compounds 20-28, 5% solutions in acetonitrile, were irradiated in asemi-micro photochemical reactor provided by Photochemical Reactors Ltd.The photochemistry of 2-nitrobenzyl esters is reported to be unchangedwithin the range 254-314 nm¹⁷ (Scheme 3). Irradiation was carried out at254 nm, the predominant wavelength of the low pressure Hg arc providedwith the reactor. Despite the fact that a weak four-watt lamp was usedas the source of UV light, a limited photolysis (c.5%) in the2-nitrobenzyl series and a very clear photolysis (20-30%) in theα-methyl-2-nitrobenzyl series was observed. The photolysis was monitoredby HPLC. 2-Nitrosobenzaldehyde (45) was the only UV detectablephotoproduct formed in the 2-nitrobenzyl series and similarly2-nitrosoacetophenone (46) was the sole product of photolysis in theα-methyl-2-nitrobenzyl series (Scheme 3). The detector used in the HPLCapparatus cannot detect the putative carboxylic acids generated duringthe photoreaction. The estimated degree of photoconversion for esters20-24 was around 5% whereas for esters 25-28 was 20-30%.α-Phenyl-2-nitrobenzyl esters 30-33, 5% solutions in acetonitrile, werealso irradiated at 254 nm (Scheme 3). The response was much morepronounced than in the α-methyl series; each of the esters gave thesame, expected product of photolysis, 2-nitrosobenzophenone (47), in40-50% yield, and its structure was confirmed by HRMS and NMR. Thephotoreactions were very selective with only small amounts of sideproducts being detected.

Irradiation of esters 30-33 at 350 nm in the same photoreactor, using aweak four watt UV lamp, 1, 6″, black phosphor coated with peak emissionat 350 nm, also resulted in a good response. This response may beattributed to the high concentration of the esters causing completelight absorption at 350 nm as well as 254 nm. Again, each of theirradiated esters gave the same and expected products of photolysis,2-nitrosobenzophenone (47), and the corresponding carboxylic acid, in30-40% yield. The photoreactions were even more selective than thosecarried out at 254 nm with no side products being detected.

The analogous α-methyl-2-nitrobenzylesters (25-28) described above,having less light absorption at 350 nm than compounds 30-33, did notrespond to irradiation at 350 nm.

A more powerful 100 W high pressure mercury arc lamp was initially usedfor the irradiation of α-phenyl-2-nitrobenzyltrichloroacetate (33) andα-(3-methoxyphenyl)-2-nitrobenzyl-trichloroacetate (35) at 365 nm(Scheme 3). The rate of photoconversion was monitored by HPLC, TLC andUV. Depending on the concentration of solution (0.002-2% inacetonitrile) and the length of light path used (1-10 mm) the rate ofphotoconversion varied between 20% and 90% for a 5 min irradiation. Theproducts of photolysis, 2-nitrosobenzophenone (47) and3′-methoxy-2-nitrosobenzophenone (48), were isolated by HPLC and showedby mass spectroscopy to be the same as those isolated previously duringirradiations at 254 and 350 nm in the photoreactor described above.Secondary photoproducts were not observed under the conditions ofrelatively weak irradiation used in the photoreactor (4 W lamp), butwere observed when the 100 W high pressure Hg arc lamp was used forextended time.

Irradiation of a 2 mm film of 0.2% solutionα-3-methoxyphenyl-2-nitrobenzyltrichloroacetate[trichloro acetic acid(3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester] (35) with the highpressure mercury arc lamp, in a flat bottom vial, gave a nearlyquantitative photoconversion to compound 48 after 5 minutes.

Irradiation of 0.2% solutions ofα-(3-methoxyphenyl)-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (38) andα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39) inacetonitrile resulted in nearly quantitative conversions into theexpected photoproducts, 4,5-dimethoxy-3′-methoxy-2-nitrosobenzophenone(50) and 4,5-dimethoxy-2-nitrosobenzophenone (51), respectively, afterless than 4 minutes. Irradiations at lower concentrations, monitored byUV, resulted in even faster photoreactions. When a 0.004% solution ofα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39) wasirradiated, there was a quantitative conversion into the photoproductafter 30 seconds. The two esters appeared to have a similar rate ofphotoconversion but the photolysis ofα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39), quantum yieldφ˜0.14, was cleaner with less amount of polar secondary photoproducts.These properties, coupled with the reduced inner filter effect andabsorption maximum at 345 nm, make compound 39 a prime candidate for usein the microarray format.

Photolysis of 5-dimethylamino-2-nitrobenzyltrichloroacetate (41) andα-phenyl-5-dimethylamino-2-nitrobenzyltrichloroacetate (42) appeared tobe of particular promise. They have absorption maxima at 395 and 398 nm,respectively, with molar extinction coefficients of around 17000 M.cm⁻¹.The photolysis of compound 41, with no substitution at the α-position,was slower than that of the esters having an α-substitution^(16,17).Thus irradiation at 0.2% concentration, at 365 nm, gave the expectedphotoproduct, 5-dimethylamino-2-nitrosobenzaldehyde (53), in 30% yieldafter 16 min. In addition, the amount of accompanying secondaryphotoproducts, around 10%, was the highest among the esters evaluated sofar. On the other hand, irradiations at lower concentrations indicatedthat the inner filter effect was diminishing as the photolysisproceeded. This was not the case with any esters tested by us before.Preliminary illuminations of compound 41 with blue light, λ>395 nm, at0.002% concentration, carried out with the high pressure mercury arclamp equipped with appropriate filters, gave about 12% photoconversionafter 16 min. This result indicates that the development of photolabileesters whose activation would require considerably less expensivesources of visible light is feasible.

It was expected that the photolysis of compound 42 having an α-phenylsubstitution^(16,17), would be faster than that of compound 41. However,its rate of photoconversion was similar to that of compound 41.Furthermore, HPLC analysis indicated the formation of several primary orsecondary photoproducts.

Irradiations of the synthesised esters with the high pressure mercuryarc lamp and He/Cd laser showed thatα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39) gave thehighest rate of desired photoconversion with virtually no secondaryphotoproducts. This, coupled with the absorption maximum at 345 nm, makethis compound a good candidate for the use as a photoacid generator inthe microarray format.

Compounds 33 and 39 that emerged as the best photoacid generators wereassessed for their suitability to effect indirect photodetritylation.This involved their irradiations in the presence of 5′-O-dimethoxytritylprotected nucleosides and oligonucleotides in indirectphotodetritylations on solid support and in solution as well asirradiations in the presence of 5′-O-dimethoxytrityl protectednucleosides attached to commercially available controlled porosity glass(CPG).

These experiments demonstrated that complete photodetritylation ispossible even if a weak source of light was employed but irradiationtimes required were between 70 and 140 minutes. Thus, >99% yielddetritylation was achieved when 1% solution of ester 39 indichloromethane²² was irradiated for 70 minutes in the presence ofcommercially available 51′-O-DMTr-T-CPG. Successful, part manual, partautomated syntheses of DMTrTT, DMTrTTT, DMTrTTTT, DMTrTTTTT andDMTrATATA were also carried out. α-Phenyl-2-nitrobenzyltrichloroacetate(33), was employed in the manual photodetritylation step performed inthe photoreactor, whereas the condensation, oxidation and capping werecarried out on the synthesiser using appropriate automated, computercontrolled protocols.

We also determined whether the detritylation could be achieved at lowerconcentrations of trichloroacetic acid. The usual trichloroacetic acidconcentration for detritylation is 2-3% (w/v), corresponding to c.120-180 mM. The photoacid precursor used,α-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39), has amillimolar extinction coefficient of 3.0 mM.cm⁻¹ at 365 nm, the mostsuitable excitation wavelength available from a high pressure Mercuryarc lamp. The absorption at 365 nm, of a 1 mm depth of a 120-180 mMester solution is therefore 36-54. Such solutions are effectively opaqueto light at 365 nm and obviously more dilute solutions of the ester wererequired.

A series of experiments involved the measurements of detritylationkinetics from CPG and from Expedite membranes using dilutetrichloroacetic acid and photogenerated trichloroacetic acid insolution. It was established that the rates of photoconversion wereconsiderably higher whereas inner filter effects were considerably lowerfor 10-20 mM solutions of the ester in dichloromethane. 10 mMTrichloroacetic acid in dichloromethane effected complete detritylationof 5′-O-dimethoxytrityl protected nucleosides after 300 seconds. Thefindings of this study were used to develop protocols for the automatedsynthesis on a DNA synthesiser.

2.2 Measurements of Quantum Yields for Photolysis of Esters

2.2.1 Methods

Quantum yields of modest accuracy are useful for ranking the value ofthe individual members of a set 2-nitrbenzyl esters as photoacidgenerators. We devised a relatively simple method that follows the timedependent course of changes in the UV-Visible absorption spectrum of anester solution during photolysis. Wavelength values for absorptionmaxima and minima, and extinction coefficients, are given above in theExperimental Section. We assumed that the photolytic reaction has firstorder kinetics. The half-time for the reaction (t_(half)), definedoperationally as the time for taken for the spectral changes to proceedto 50% completion, is related to the first order rate constant k by theequation k=0.693/t_(half) sec⁻¹.

The initial rate of photolysis at the onset of irradiation is theproduct of k and the amount of ester in the illuminated cuvette. Therate of light absorption at the onset of irradiation is the product ofthe incident light power and the fractional absorption of light by thesolution. The later value is equal to 1-10_(−A), where A is theabsorption of the ester solution at the excitation wavelength prior toillumination. The quantum yield Φ is defined as the ratio of the rate ofphotolysis, expressed as molecules per second, to the rate of lightabsorption expressed as photons per second.

If the photolysis rate is expressed as μmoles/s and the light absorptionrate as mJ/sec, then the quantum yield can be conveniently calculated asQuantum yield=(photolysis rate)/(light absorption rate)×(a wavelengthdependent constant). This constant incorporates the appropriatemolecular and optical conversion factors and has a value of 3.38×10² at365 nm, and 2.96×10² at 405 nm. The half-life itself can be obtainedeither from the slope of a plot of log (fraction of ester unphotolysed)versus time, or more rapidly but less accurately by inspection of a setof absorption spectra recorded during photolysis and including both zeroand full photolysis.

2.2.2 Results

The Table summarizes our results using the above methods as applied toseveral esters. All excitations were at 365 nm. Solutions of esters wereeither at low (50-150 μM) or substantially higher (5.4-5-6 mMconcentration. In the later cases all incident light is absorbed, andspectroscopic changes were followed by dilution of intermittent samplesinto 3.0 ml of solvent (dichloromethane). The ranges given for thequantum yield are those for 2-3 separate determinations.

TABLE Quantum yields for photolysis of several esters at 365 nm Ester Φα-Phenyl-5-dimethylamino-2- <0.01 nitrobenzyltrichloracetate (41)α-Phenyl-5-dimethylamino-2,4- 0.003-0.005 dinitrobenzyltrichloracetate(44) α-Phenyl-5-dimethylamino-2,6- 0.04-0.08dinitrobenzyltrichloracetate (43) α-Phenyl-4,5-dimethoxy-2- 0.12-0.15nitrobenzyltrichloracetate (39) α-Phenyl-4,5-dimethoxy-2,6- 0.43-0.46dinitrobenzyltrichloracetate (40)2.3 Experimental5 2.3.1 Irradiations of Photolabile Esters in the Photoreactor

Compounds 20-44 were irradiated in a semi-micro photochemical reactorprovided by Photochemical Reactors Ltd. Irradiations were carried outusing a four watt UV 10 lamp with peak emission at 254 nm or 350 nm. Theprogress of photolysis was monitored by HPLC and TLC. Reverse phase HPLCwas performed using a Waters chromatography system with a variablewavelength detector set at 254 nm and 280 nm. Columns, Waters Delta Pak5 μ C18-300A, were 15 used for analytical and preparative scales. Themobile phases were (A) 0.05 M aq. [Et₃NH]⁺[CH₃COO]⁻(B) MeCN. Gradientelution; 5%(B)-90% (B) over 30 minutes.

Irradiation of α-phenyl-2-nitrobenzyltrichloroacetate [trichloro-aceticacid (2-nitro-phenyl)-phenyl-methyl ester] (33)

α-Phenyl-2-nitrobenzyltrichloroacetate (33), 5% (w/v) solution inacetonitrile (3.5 mL), in a 1 cm quartz cuvette, was irradiated in thephotoreactor at 350 nm for 70 minutes. The irradiated solution wasanalysed by HPLC showing the presence of the starting material,retention time R_(t)=18.33 min, and the photoproduct, R_(t)=15.15 min.The estimated degree of photoconversion, peak areas, was 15%.Preparative HPLC resulted in two fractions, fraction 1, retention time15.15 min fraction 2, retention time 18.33 min. Each fraction wasanalysed by MS; the photoproduct, 2-nitrosobenzophenone (47), was foundin fraction 1; (M+H) 212.1.

Similarly, esters 22-44 were irradiated in the photoreactor at 254 nmand 350 nm giving a varying degree of photoconversion. Each photoproduct(45-56) was isolated by preparative HPLC, estimated yield in brackets,and its identity was confirmed by mass spectroscopy; (M+H) values givenin brackets.

Acetic acid 2-nitro-benzyl ester (20): R_(t)13.67 min

Trimethyl-acetic acid 2-nitro-benzyl ester (21): R_(t) not determined

Chloro-acetic acid 2-nitro-benzyl ester (22): R_(t) 15.44 minDichloro-acetic acid 2-nitro-benzyl ester (23): R_(t) not determined.

Trichloro-acetic acid 2-nitro-benzyl ester (24): R_(t) not determined.

Acetic acid 1-(2-nitro-phenyl)-ethyl ester (25): R_(t) 14.57 min.

Chloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (26): R_(t) 15.44 min.

Dichloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (27): R_(t) 16.40min.

Trichloro-acetic acid 1-(2-nitro-phenyl)-ethyl ester (28): R_(t) 17.24min.

Trimethyl-acetic acid 1-(2-nitro-phenyl)-ethyl ester (29): R_(t) notdetermined.

Acetic acid (2-nitro-phenyl)-phenyl-methyl ester (30): R_(t) 15.88 min.

Chloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (31): R_(t)16.48 min.

Dichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (32): R_(t)17.20 min.

Trichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester (33): R_(t)18.33 min.

Chloro-acetic acid (3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(34):R_(t) not determined.

Trichloro-acetic acid (3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(35):R_(t) 16.77 min.

Dichloro-acetic acid (4-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(36): R_(t) 16.22 min.

Trichloro-acetic acid (4-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(37): R_(t) not determined.

2-Nitrosobenzaldehyde (45)²³: R_(t) 12.08 min (M+H) 135.1 (5-7% at 254nm).

2-Nitrosoacetophenone (46)²⁴: R_(t) 12.25 min; (M+H) 150.1 (20-30% at254 nm).

2-Nitrosobenzophenone (47)²⁵: R_(t) 15.15 min; (M+H) 212.1 (40-50% at254 nm; 30-40% at 350 nm); observed FAB MS 212.0701, [C₁₃H₁₀NO₂+H]⁺requires 212.0712; ¹³C-NMR δ 120.26 (C-3), 127.87 (C-3′, C-5′), 128.78(C-2′, C-6′), 129.15 (C-1) 129.34 (C-4), 129.41 (C-4′), 131.59 (C-5),136.86 (C-1′), 163.91 (C-2), 195.94 (CO).

3′-Methoxy-2-nitrosobenzophenone (48): R_(t)14.11 min; (M+H) 242.1(30-40% at 350 nm) observed FAB MS 242.0828, [C₁₅H₁₄NO₄+H]⁺ requires242.0817.

4′-Methoxy-2-nitrosobenzophenone (49): R_(t) 14.22 min; (M+H) 242.1 50%.

Preparative scale irradiation ofα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate[trichloro-aceticacid (4,5-dimethoxy-2-nitro-phenyl)-phenyl-methyl ester] (39),α-phenyl-4,5-dimethoxy -2,6-dinitrobenzyltrichloroacetate[trichloro-acetic acid (4,5-dimethoxy-2,6-dinitro-phenyl)-phenyl-methylester] (40) and α-phenyl-5-dimethylamino-2,6-dinitrobenzyltrichloroacetate [trichloro-acetic acid(5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methyl ester] (43)

Esters 39 and 40, 2% solution in dichloromethane (3.5 mL), and ester 43,0.25% solution in dichloromethane (3.5 mL), were irradiated in thephotoreactor at 350 nm for 200 minutes. The degree of photoconversioncould be conveniently monitored by TLC. Each solution was concentratedto a half of its volume and applied onto a column of silicagel (4 g,Coarse silicagel). The column was eluted with CH₂Cl₂, appropriatefractions were combined and the solvent was removed in vacuo. Theresidue was dissolved in water/ethanol (1:1) (2 mL) and freeze-dried togive the photoproduct.

4,5-Dimethoxy-2-nitrosobenzophenone (51); yield 62%; Rf (CH₂Cl₂,) 0.58;green-yellow froth, observed FAB MS 272.0920, [C₁₅H₁₄NO₄+H]⁺ requires272.0923; ¹H-NMR δ 3.89 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 6.74 (s, 1H,H-3), 7.44 (s, 1H, H-6), 7.54 (m, 2H, H-3′, H-5′), 7.63 (m, 1H, H-4′)7.75 (m, 2H, H-2′, H-6′); ¹³C-NMR δ 56.37(OCH₃), 57.38 (OCH₃),95.32(C-3), 110.39(C-6), 129.15 (C-3′, C-5′), 129.64 (C-2′, C-6′),134.13 (C-4′) 137.18 (C-1), 138.06 (C-1′), 150.36 (C-5), 156.82 (C-4),160.78 (C-2), 195.99 (CO); UV λ_(max) 385 nm ε 8762.

4,5-Dimethoxy-6-nitro-2-nitrosobenzophenone (52); yield 69%; Rf(CH₂Cl₂,) 0.64; yellow froth, observed FAB MS 317.0778, [C₁₅H₁₃N₂O₆+H]⁺requires 317.0774; ¹H-NMR δ 3.89 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 6.74(s, 1H, H-3), 7.44 (s, 1H, H-6), 7.54 (m, 2H, H-3′, H-5′), 7.63 (m, 1H,H-4′), 7.75 (m, 2H, H-2′, H-6′); ¹³C-NMR δ 57.80 (OCH₃), 63.10 (OCH₃),101.31 (C-3), 126.38 (C-1), 126.55 (C-4′), 129.39 (C-3′, C-5′), 129.48(C-2′, C-6′), 134.92 (C-4′), 137.39 (C-1′), 148.21 (C-5), 154.65 (C-4),158.36 (C-2), 191.85 (CO); UV λ_(max) 372 nm ε 4433.

5-Dimethylamino-6-nitro-2-nitrosobenzophenone (55); yield 47%; Rf(CH₂Cl₂,) 0.66; yellow solid, mp indef; ¹H-NMR δ 2.90 [s, 6H, N(CH₃)₂],6.28 (d, 1H, J=8.72 Hz, H-4), 7.48 (m, 5H, H-2′-H-6′), 8.25 (d, 1H,J=8.72 Hz, H-3); ¹³C-NMR δ 43.50 [N(CH₃)₂], 98.96 (C-4), 107.78 (C-3),126.96 (C-1), 128.04 (C-3′, C-5′), 129.29 (C-4′), 129.95 (C-2′, C-6′),130.40 (C-1′), 132.77 (C-6), 144.97 (C-5), 151.70 (C-2), 166.62 (CO); UVλ_(max) 447 nm ε 20550.

Irradiation of α-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate[trichloro-acetic acid (4,5-dimethoxy-2-nitro-phenyl)-phenyl-methylester] (39) in the presence of 5′-O-dimethoxytritylthymidine bound tocontrolled porosity glass (5′-O-DMTr-T-CPG).

Commercially available 5′-O-DMTr-T-CPG (50 mg with a loading of 35μmole/g) was treated with 2% solution of ester 31 in dichloromethane(3.5 mL). The mixture was irradiated at 350 nm for 70 minutes. Thesolution turned orange. The solid was filtered off, washed thoroughlywith dichloromethane and dried in a desiccator over P₂O₅. A weighedsample of the solid was treated with a measured amount of 3%trichloroacetic acid in dichloromethane and subsequently absorption at494 nm was measured indicating that the detritylation occurred in>90%yield.

2.3.2 Irradiations of Photolabile Esters using 100 W High PressureMercury Arc Lamp

A more powerful and intense source of ultraviolet irradiation wasprovided by a 100 W high pressure Hg arc lamp (Osram HBO 100 W/2)in aPhoton Technology International (“PTI”) f/4.5 ellipsoidal reflector unitmodel A-1010B, and powered with an LPS-220B supply (also from PTI).Cooling was by water circulation with a closed cycle cooling box fromOn-Line Instruments Systems (“OLIS”). The reflected beam from the lampwas passed through a liquid heat filter (Oriel Instruments Model 6123)that provides an 80 mm optical path length through water. The beam wasthen passed through the following Schott filters, each 3 mm thick: KG1(further heat removal), WG320 (removal of UV light below 320 nm) and UG1(transmission of UV-light between 310 and 390 nm). The final outputconsisted therefore of predominantly the 365 nm Hg arc line, with a muchsmaller contribution from the 334 nm line. The output was then focussedwith a suitable lens or lenses to the face of a stoppered quartzcuvette, volume 4.0 ml and path length 1 cm.The contents of the cuvettewere stirred at c.5 Hz with an 8×3 mm magnetic stirring bar, driventhrough a local drive unit and remote control unit (Variomag Model MINI,H+P Labortechnik GmBH).

The light power falling on the cuvette was measured with a Melles GriotBroadband 2 W Power/Energy Meter model 13 PEM001. The detector, whichhas a 10 mm diameter thermopile, was positioned to collect light passingthrough the cuvette position, and the power measured in the of thecuvette's absence was equated with the incident power in its presence.The output of the Hg arc measured at the cuvette position fell with lampage, from about 120 mW for a new lamp to about 30 mW for a used lampabout to fail. Attenuation of these incident powers was achieved whenrequired with changes of beam focussing and/or the introduction of ametal mask with a hole immediately prior to the cuvette. A mechanicalshutter was used to achieve complete attenuation.

All exposures of solutions to UV irradiation were carried out at roomtemperature in the range 18-22° C. Absorption spectra of the irradiatedsolutions were obtained by transferring the cuvette and its contents toa Beckman Model DU-7 spectrophotometer and measuring against a solventblank. Alternatively, when the peak absorption of the contents was high(>1.5), a sample was taken from the cuvette with a micro syringe anddiluted into a final volume of 3.0 ml of solvent in another cuvette onwhich the measurements were made.

The progress of photolysis was monitored by HPLC and TLC. Reverse phaseHPLC was performed using a Waters chromatography system with a variablewavelength detector set at 254 nm and 280 nm. Columns, Waters Delta Pak5μ C18-300A, were used for analytical and preparative scales. Unlessotherwise indicated the mobile phases were (A) 0.05 M aq.[Et₃NH]⁺[CH₃COO]⁻ (B) MeCN. Gradient elution 5% B-90% B over 30 minutes.

Compounds 33, 35, 38-44 were irradiated in a 1 mm quartz cuvette using a100 W high-pressure mercury arc lamp.

Irradiation of α-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate[trichloro-acetic acid (2-nitro-phenyl)-phenyl-methyl ester] (39)

α-Phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39), 0.2% (w/v)solution in acetonitrile in a 1 mm quartz cuvette, was irradiated at 365nm. Samples were taken at regular, 30 sec, intervals and analysed byHPLC showing the presence of the starting material, retention timeR_(t)=16.68 min, and the photoproduct, R_(t)=13.45. The estimated degreeof photoconversion after 240 seconds was 90%. Preparative HPLC resultedin two fractions, fraction 1, retention time 16.68 min and fraction 2,retention time 13.45 min. Each fraction was analysed by MS; thephotoproduct, 4,5-dimethoxy-2-nitrosobenzophenone (51), was found infraction 1; (M+H) 272.1.

Similarly, esters 33, 35, 38, 40, 41,42,43 and 44 were irradiated in a 1mm quartz cuvette at 365 nm giving a varying degree of photoconversion.Each photoproduct (47, 48, 50-56) was isolated by preparative HPLC,estimated yield in brackets, and its identity was confirmed by massspectroscopy; (M+H) values given where appropriate. Trichloro-aceticacid (2-nitro-phenyl)-phenyl-methyl ester (33): R_(t) 18.33 min.

Trichloro-acetic acid (3-methoxy-phenyl)-(2-nitro-phenyl)-methyl ester(35): R_(t) 16.77 min.

Trichloro-acetic acid(4,5-dimethoxy-2-nitrophenyl)-(3-methoxyphenyl)-methyl ester (38): R_(t)17.02 min.

Trichloro-acetic acid (4,5-dimethoxy-2-nitrophenyl)-phenyl-methyl ester(39): R_(t) 16.63 min.

Trichloro-acetic acid (4,5-dimethoxy-2,6-dinitrophenyl)-phenyl-methylester (40): R_(t) not determined.

Trichloro-acetic acid (5-dimethylamino-2-nitro-phenyl)-phenyl-methylester (42): R_(t) 17.62 min.

Trichloro-acetic acid (5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methylester (43): R_(t) 14.42 min (A) 0.05M aq. [Et₃NH]⁺[HCO₃]⁻(B) MeCN.Gradient elution over 30 minutes.

Trichloro-acetic acid (5-dimethylamino-2,4-dinitro-phenyl)-phenyl-methylester (44): R_(t) not determined 2-Nitrosobenzophenone (47): R_(t) 15.15min; (M+H) 212.1 (90%, 300 seconds).

3′-Methoxy-2-nitrosobenzophenone (48): R_(t)14.11 min; (M+H) 242.1 (80%,240 seconds, TLC).

3′-Methoxy-4,5-dimethoxy-2-nitrosobenzophenone (50): R_(t) 14.25 min;(M+H) 302.0 (23%, 120 seconds).

4,5-Dimethoxy-2-nitrosobenzophenone (51): R_(t) 13.45 min; (M+H) 272.0(90%, 240 seconds).

4,5-Dimethoxy-6-nitro-2-nitrosobenzophenone (52): R_(t) 13.45 min; (M+H)317.1.

5-Dimethylamino-2-nitrosobenzaldehyde (53)²⁶: R_(t) 15.83 min; (M+H)179.2 (30%, 16 min)

5-Dimethylamino-2-nitrosobenzophenone (54): R_(t) 16.87 min; (M+H)255.1.

5-Dimethylamino-2-nitro-6-nitrosobenzophenone (55): R_(t) 14.77 min (A);(M+H) 300.3 0.05M aq. [Et₃NH]⁺ [HCO₃]⁻ (B) MeCN. Gradient elution over30 minutes. (70%, 45 minutes TLC).

5-Dimethylamino-4-nitro-2-nitrosobenzophenone (56): R_(t) notdetermined; (M+H) 300.3.

Irradiation of α-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate[trichloro-acetic acid (4,5-dimethoxy-2-nitro-phenyl)-phenyl-methylester] (39) in the presence of 5′-O-dimethoxytritylthymidine bound tocontrolled porosity glass (5′-O-DMTr-T-CPG)

Commercially available 5′-O-DMTr-T-CPG (35 μmol/g) (5 mg) was treatedwith 1% (w/v) solution of ester 39 in dichloromethane (4.0 mL). Themixture was irradiated at 365 nm for 5 minutes. The solution turnedorange. The solid was filtered off, washed thoroughly withdichloromethane and dried in a desiccator over P₂O₅. A weighed sample ofthe solid was treated with a measured amount of 3% trichloroacetic acidin dichloromethane. Subsequently, absorption at 494 nm was measuredindicating that the detritylation occurred in>99% yield.

Conclusion: Photogenerated trichloroacetic acid in dichloromethane at10-16 mM gives>99% detritylation of DMT-T.

Preparative scale irradiation ofα-phenyl-5-dimethylamino-2,6-dinitrobenzyltrichloroacetate[trichloro-acetic acid(5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methyl ester] (43) andα-phenyl-5-dimethylamino-2,4-dinitrobenzyltrichloroacetate[trichloro-acetic acid(5-dimethylamino-2,4-dinitro-phenyl)-phenyl-methyl ester] (44)

Esters 43 and 44, 0.25% solution in dichloromethane (3.5 mL), wereirradiated in the photoreactor at 350 nm for 200 minutes. The degree ofphotoconversion could be conveniently monitored by TLC. Each solutionwas concentrated to a half of its volume and applied onto a column ofsilicagel (4 g, coarse silicagel). The column was eluted with CH₂Cl₂,appropriate fractions were combined and the solvent was removed invacuo. The residue was dissolved in water/ethanol (1:1) (2 mL) andfreeze-dried to give the photoproduct.

5-Dimethylamino-2-nitroso-6-nitrobenzophenone (55); yield 58%;spectroscopic data were consistent with those quoted above for the samecompound.

5-Dimethylamino-2-nitroso-4-nitrobenzophenone (56); yield 27%; Rf(CH₂Cl₂,) 0.45; yellowsolid; [M+H]⁺300.2; ¹H-NMR δ 3.11 [s, 6H,N(CH₃)₂], 7.18 (s, H-6, 7.25-7.76 (m, 5H, H-2′-H-6′), 8.03 (s, 1H, H-3).

2.3.3 Titration of Acid Production from the Photolysis ofα-phenyl-4,5-dimethoxy-2-nitrobenzyl trichloroacetate [trichloro-aceticacid (4,5-dimethoxy-2-nitro-phenyl)-phenyl-methyl ester] (39) usingtetrabromophenol-blue as indicator

Acid production from photolysis of4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39) was measured bytitration with alkali and a visual indicator (tetrabromophenol blue, pKc.3.5. Aldrich).

Irradiation was performed using a 100 W water-cooled high pressure Hgarc, Schott glass filters to isolate a broad band from 330 to 380 nm(KG12 heat filter, WG320 blocking below 320 nm, and UG1, all 3 mmthick.). Glass cuvettes, 1 cm light path, volume 4.0 mL, were used asreaction vessels. Starting concentrations of the ester in 3.1 mL ofacetonitrile ranged from 19 to 222×10⁻⁶M.

Illumination time was 2 min for ester concentrations less than148×10⁻⁶M, otherwise 3 min. Samples were stirred during illumination byhand with a plastic stirrer rod. After illumination, tetrabromophenolblue, sodium salt, was added to a concentration of 22×10⁻⁶ M. The bluecolour of the indicator disappeared except for the controls lackingeither ester or irradiation. Standardised solutions of sodium methoxidewere then added to the cuvettes, with stirring, until the indicatorbecame blue/green.

Results: The amount of acid required to restore the blue/green colour ofindicator added to irradiated cuvettes was proportional to the startingconcentration of ester. The slope of the plot was 0.92, close to thetheoretical value of 1.0.

Conclusion: The results clearly identify the formation of acid inamounts close to the expected amount from ester photolysis.

SUMMARY

Photolytic acid production fromα-phenyl-4,5-dimethoxy-2-nitrobenzyl-trichloroacetate (39) was measuredby titration of UV-irradiated solutions with sodium methoxide solutions,using tetrabromophenol blue (pK 3.5) as indicator. Irradiation times,illumination geometry and internal filter effect made for non-optimalconditions, and possibly led to underestimate of the degree ofphotolysis. Nevertheless, the amount of acid produced was proportionalto the amount of ester photolysed over a range of initial esterconcentrations. The molar ratio of acid produced to ester photolysed was0.92, in reasonable agreement with the theoretical value of 1.0.

2.3.4 Photodetritylation usingα-phenyl-5-dimethylamino-2,6-dinitrobenzyltrichloracetate (43)

Experimental Design

The experiment takes advantage of the strong visible absorption band ofthe dimethoxytrityl cation (DMTr⁺) in acid (λ_(max)=498 nm, E_(mM)=80).However, the presence of a large excess of the photoproduct(s) of 43interferes with absorption measurement of DMTr⁺ in the 450-520 nmregion. An indirect approach was therefore taken in which the amount ofDMtr⁻ remaining on cpg after exposure to a solution of illuminated 43was measured after separation of the cpg particles by centrifugationand, following a wash and further centrifugation to remove residualcontamination by supernatant, addition of 3% trichloroacetic acid to theparticles. The added trichloroacetic acid rapidly detritylates residualDMTr-T-cpg, releasing DMTr⁺. The absorbance of the suspension of cpg inacid at 498 nm therefore accurately reflects the amount of DMTr that wasattached to the cpg particles after exposure to a solution of 43 with orwithout exposure to UV light.

2.3.4.1. Experimental Procedure and Results

Dichloromethane was used for all solutions and washes. DMTrT-cpgparticles(accurately measured to be within ±3% of 5 mg dry weight) wereadded to 3.0 ml of dichloromethane containing 2.54 mg of 43 per mL in a4.0 ml quartz cuvette of path length 1 cm. A small magnetic stirring barwas added, and the cuvette was sealed with its stopper. The cuvette wasplaced in the output beam of the Hg ars lamp, as described above under“Instruments” and exposed to UV light power of 108 mW for 20 minutes.Absorption spectra from 300-600 nm of 100-fold diluted samples of thecuvette contents before and after irradiation confirmed that extensivephotolysis had occurred in that time, as evidenced by the fall inabsorption of the main peak of the ester at 376 nm and the emergence ofthe new peak of the major photoproduct at 450 nm.

The cpg particles of each sample (i.e. irradiated test andnon-irradiated control) were separated from their supernatants bytransferring them to glass centrifuge tubes with 35 ml ofdichloromethane and briefly centrifuged. The supernatant was removed.After a further wash the particles were taken up in 5.0 ml of 3% (w/v)trichloroacetic acid. The control particles gave an intense orangecolour whereas the test particles gave no detectable colour.Spectrophotometric measurement showed the expected peak at 498 nm(absorption value>3.0), whereas the test sample showed no increase overa solvent baseline at an absorption detection limit of 0.005.

Conclusion: these data demonstrate that DMTt-T-cpg present during thephotolysis of 43 became at least 99.9% detritylated.

3. Automated Photodirected Synthesis of Oligonucleotides

3.1. Automated Photodirected Oligonucleotide Synthesis on the DNASynthesiser using a High Pressure Mercury Arc Lamp with a Flexible LightGuide

A high-pressure mercury arc lamp with an UV conducting liquid lightguide, suitable for attaching to the automated oligonucleotidesynthesiser, was used to irradiate solutions of the esters being passedthrough modified flow columns housing controlled porosity glass (CPG).The automated photodirected syntheses of a control pentamer, DMTrTTTTT,demonstrated that the best results were obtained when 300 pulses, around4.5 mL, of a 1% (22 mM)(solution ofα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39) indichloromethane were passed over 1800 seconds through the irradiatedmodified flow column, housing 0.5 μmole of CPG, during eachdetritylation step. This ensured sufficient steady state concentrationsof photogenerated trichloroacetic acid (2-4 mM) as well as adequatecontinued flow during photolysis on the synthesiser to avoid the effectsof unstirred and unilluminated volumes. As estimated by MS and HPLC theoverall yield was at least 81.8% which corresponded to 95.1% stepwiseyield. It was found that a similar yield was obtained when the pentamerwas made using the conventional synthetic protocol with 3%trichloroacetic acid in dichloromethane. The same protocol was employedin the synthesis of a variety of sequences which included DMTrTTTTT,DMTrTATAT DMTrTGTGT, DMTrTTTTTTTTTTT, DMTrATATATATAT, DMTrCTCTCTCTCT andDMTr TGCATTGCAT and the yield of desired products was essentially thesame irrespective of whether trichloroacetic acid was added directly at183 mM concentration or generated photochemically from 22 mM precursor.

For example, the estimated stepwise yield for synthesis ofDMTrTTTTTTTTTT, in our hands was 97.8% for the photochemical method and98% for the conventional method. The yield for a TT coupling using theconventional method is generally considered to be in the region of98-99%, so we have little if any room for improvement. Couplingefficiencies involving purine nucleosides, particularly guanosine, areusually lower. Overall yields, using either conventional orphotodirected methods, depend strongly on the performance of a DNAsynthesiser used. This is reflected in the total and stepwise yieldsobtained by us and presented in Table 5.

These results clearly demonstrate that detritylation performed usingphotogenerated trichloroacetic acid is as effective as that achieved byconventional 183 mM trichloroacetic acid in dichloromethane. The nitrosophotoproduct does not appear to block the newly exposed 5′-OH group ofthe oligonucleotides. To our knowledge no other group has achievedequality of yield between photodirected and conventional syntheses. Twogroups, at Affymetrix and Houston, using photoacid generators availablefrom the semiconductor industry, fell several % Short^(27,28,29).

The use of α-phenyl-4,5-dimethoxy-2,6-dinitrobenzyltrichloroacetate (40)instead of α-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39),also resulted in equality of yield between photodirected andconventional syntheses. The high quantum yield and increased rate ofphotolysis of compound 40, highlighted earlier, enabled not only the useof its more dilute solutions but also considerable shortening ofsynthetic cycles. The automated photodirected syntheses of a controlpentamer, DMTrTTTTT, demonstrated that the best results were obtainedwhen 150 pulses, around 2.2 mL, of a 0.5% (11 mM) solution of compound40 in dichloromethane were passed over 900 seconds through theirradiated modified flow column, housing 0.5 μmole of CPG, during eachdetritylation step. This improved and shortened protocol was employed inthe synthesis of a variety of sequences which included DMTrTTTTT,DMTrTATAT DMTrTGTGT, DMTrTTTTTTTTTTT, DMTrATATATATAT, DMTrCTCTCTCTCT,DMTrGTGTGTGTGT and DMTrTGCATTGCAT and the results are presented in Table8.

Like its counterpart 51 described above,4,5-dimethoxy-6-nitro-2-nitrosobenzophenone (52), the photoproductformed as a result of the irradiation ofα-phenyl-4,5-dimethoxy-2,6-dinitrobenzyltrichloroacetate (40) does notappear to block the newly exposed 5′-OH group of the oligonucleotides.

3.2. Experimental

We used a Millipore Expedite DNA synthesiser with modified polypropyleneflow columns and provision of a shuttered UV source (365 nm line from a100 W high pressure Hg arc, from Linos) to illuminate the column througha flexible light guide and suitable optical filters and lenses usingeither α-phenyl-2-nitrobenzyltrichloroacetate (39), orα-phenyl-2,6-dinitrobenzyltrichloroacetate (40), in place of theconventional direct addition of trichloroacetic acid. Three hundredpulses, (4.5 mL), of a 1% (w/v) (22 mM) solution ofα-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetate (39) indichloromethane were passed over 1800 seconds through the irradiatedmodified flow column, housing 0.5 μmole of 5′-O-DMTr-T-CPG (controlledporosity glass), during each detritylation step whereas the same stepperformed using α-phenyl-4,5-dimethoxy-2,6-dinitrobenzyltrichloroacetate(40) required one hundred fifty pulses, (2.2 mL), of its 0.5% (w/v) (11mM) solution in dichloromethane passed over 900 seconds through theirradiated modified flow column, housing 0.5 μmole of 5′-O-DMTr-T-CPG(controlled porosity glass). Otherwise conditions were as usual, withconventional computer controlled DNA 1 micromole protocols for capping,oxidation, coupling and washing.

Each photodirected synthesis was followed by a control, conventionalsynthesis in which 3% trichloroacetic acid in dichloromethane was usedinstead of the photoacid generator. After the completion of eachsynthesis the solid support was treated with concentrated ammoniumhydroxide (2 mL) for 18 h at room temperature. The solid was filteredoff and concentrated in vacuo on the Speedvac rotary concentrator.

Each residue was analysed by reverse phase HPLC. The reverse phase HPLCwas performed using a Waters chromatography system with a variablewavelength detector set at 254 nm and 280 nm. Columns, Waters Delta Pak5μ C18-300A, were used for analytical and preparative scales. Unlessotherwise indicated the mobile phases were A) 0.05M aq. [Et₃NH]⁺[CH₃COO]⁻ B) MeCN. Gradient elution; 5% B -60% B over 30 minutes.

Each analytical HPLC was followed by a preparative HPLC; collectedfractions were freeze-dried and analysed by electro-spray massspectroscopy. Yields, retention times and mass spectra of synthesisedtrityl-on oligonucleotides using ester 39 are presented in Tables 5 and6. Subsequent detritylation of purified sequences obtained using ester39 was carried out with 3% aqueous acetic acid over 12-15 min at roomtemperature (22-25°). The same reverse phase columns were used foranalytical and preparative HPLC of the detritylated sequences. Themobile phases were A) 0.05M aq. [Et₃NH]⁺[HCO3]⁻B) MeCN. Gradientelution; 5% B-60% B over 30 minutes. Retention times and mass spectra ofsynthesised trityl-off oligonucleotides are presented in Table 7.Yields, retention times and mass spectra of synthesised trityl-onoligonucleotides using ester 40 are presented in Tables 8 and 9.

TABLE 1 Actions in the photodirected synthesis of an oligonucleotidearray. Steps 1 to 4 constitute a synthetic cycle. They must occur 4times to extend the array by 1 base, and 4N times to make an array ofN-mers. A, C, G & T refer to the nucleoside bases. Stage Action StartGlass surface with attached linkers. The terminal —OH group is blockedwith a photosensitive group, as are the 5′-OH groups of subsequentlyadded nucleoside phoshoramidites. Step 1 Deblock the protected —OH atselected elements by patterned illumination Step 2 Couple 5′-O-protectednucleoside phosphoramidite A to free —OH groups Step 3 Cap non-reacted—OH groups with acetic anhydride Step 4 Oxidise the trivalent phosphitebond to pentavalent phosphate Step 5 Return to Step 1 and repeat in turnfor monomers C, G & T. Step6 Continue by repeating steps 1 to 5 for N-1times to make an array of N-mers Finish Deprotect exocyclic N-groups

TABLE 2 Illumination sequence during extension of an oligonucleotidearray from length n to n + 1. Synthetic Added Illumination at elementsfor A, C, G or T- cycle no. base A C G T 1 A Scheduled Stray Stray Stray2 C Stray Scheduled Stray Stray 3 T Stray Stray Scheduled Stray 4 GStray Stray Stray Scheduled

TABLE 3 Exposure time and photolysis by scheduled and stray light. Theexposure time is in units of the half- time for photolysis by scheduledlight. The calculations use equations (1) and (2) for scheduled lightand stray light respectively, and a contrast ratio of 400:1. Exposuretime 0 0.0625 0.125 0.25 0.5 1 2 4 8 16 (half-times) Scheduled 0 4.248.30 15.9 29.3 50.0 75.0 93.8 99.6 100 photolysis (%) Stray light 00.011 0.022 0.043 0.086 0.17 0.35 0.69 1.4 2.7 photolysis (%)

TABLE 4 Calculated effect of stray light at different contrast ratios onthe % of sequences identical to the designed sequences by photodirectedsynthesis of a 20-mer array using direct photodeprotection for 10half-lives. Contrast ratio 1 × 10⁴ 4 × 10³ 2 × 10³ 1 × 10³ 4 × 10² 2 ×10² 1 × 10² % of correct 96 90 81 66 35 13 1.6 sequences

TABLE 5 Yields of Oligonucleotides Obtained by Conventional orPhotodirected Synthesis; Ester 39 Synthetic Yields of Ratio ofOligonucleotides (%) stepwise Photodirected Conventional yields:Oligonucleotide Step- Step- Photodirected (DMTr-) Overall wise Overallwise Conventional -(T)₅ 81.8 95.1 75.9 93.3 1.019 -TATAT 65.2 89.9 64.989.8 1.001 -TGTGT 59.3 87.8 56.1 86.5 1.016 -(T)₁₀ 82.0 97.8 83.7 98.00.997 -(TA)₅ 51.7 92.1 51.9 93.0 0.990 -(TC)₅ 59.1 94.3 58.3 94.2 1.001-(TGCAT)₂ 60.1 94.5 65.0 95.3 0.992

TABLE 6 Retention Times and Mass Spectra of SynthesisedOligonucleotides; Ester 39 Retention Retention Molecular Time MolecularTime Ion (min). Ion Observed. (min). Observed. OligonucleotidePhotodirected Photodirected Conventional Conventional DMTrTTTTT 13.351759.6 [M − H]⁻ 13.32 1759.6 DMTrTATAT 13.10 1777.3 [M − H]⁻ 13.071777.3 DMTrTGTGT* 12.75 1809.3 [M − H]⁻ 12.70 1809.3 DMTrTTTTTTTTTT12.92 1639.6 [M − 2H]²⁻ 12.85 1639.6 DMTrTATATATATA 12.25 1662.4 [M −2H]²⁻ 12.22 1662.4 DMTrTCTCTCTCTC 12.30 1602.1 [M − 2H]²⁻ 12.27 1602.1DMTrTGCATTGCAT* 12.25 1658.6 [M − 2H]²⁻ 12.27 1658.5 *0.05 Mtriethylammonium bicarbonate buffer/acetonitrile

TABLE 7 Retention Times and Mass Spectra of SynthesisedOligonucleotides; Ester 39 Retention Molecular Retention MolecularOligonucleotide Time (min). Ion Observed. Time (min). Ion Observed(5′-3′) Photodirected Photodirected Conventional Conventional TTTTT 8.781457.2 [M − H]⁻ 8.80 1457.3 [M − H]⁻ TATAT 7.75 1475.3 [M − H]⁻ 7.831475.4 [M − H]⁻ TGTGT 7.07 1507.2 [M − H]⁻ 7.12 1507.3 [M − H]⁻TTTTTTTTTT 7.87 1488.5 [M − 2H]²⁻ 7.90 1488.7 [M − 2H]²⁻ ATATATATAT 7.901511.0 [M − 2H]²⁻ 7.92 1511.2 [M − 2H]²⁻ CTCTCTCTCT 7.63 1450.9 [M −2H]²⁻ 7.58 1450.8 [M − 2H]²⁻ TACGTTACGT 7.33 1507.6 [M − 2H]²⁻ 7.351507.5 [M − 2H]²⁻

TABLE 8 Yields of Synthesised Oligonucleotides; Ester 40 SyntheticYields of Ratio of Oligonucleotides (%) stepwise PhotodirectedConventional yields: Oligonucleotide Step- Step- Photodirected (DMTr-)Overall wise Overall wise Conventional -(T)₅ 79.5 94.4 82.3 95.2 0.991-TGTGT 76.7 93.6 79.8 94.5 0.990 -(T)₁₀ 79.1 97.4 80.6 97.6 0.998 -(AT)₅65.0 95.3 62.2 94.9 1.004 -(CT)₅ 73.4 96.6 72.3 96.4 1.002 -(GT)₅ 67.595.7 69.6 96.0 0.997 -(TGCAT)₂ 54.1 93.4 58.8 94.3 0.990

TABLE 9 Retention Times and Mass Spectra of SynthesisedOligonucleotides; Ester 40 Retention Retention Time* Molecular Time*Molecular Oligonucleotide (min). Ion Observed. (min). Ion Observed.DMTr- Photodirected Photodirected Conventional Conventional (T)₅ 13.371759.4 [M − H]⁻ 13.37 1759.4 TGTGT 12.77 1809.3 [M − H]⁻ 12.78 1809.4(T)₁₀ 12.85 1639.7 [M − 2H]²⁻ 12.90 1639.7 (AT)₅ 12.33 1662.4 [M − 2H]²⁻12.30 1662.0 (CT)₅ 12.25 1602.7 [M − 2H]²⁻ 12.30 1602.9 (GT)₅ 11.981702.0 [M − 2H]²⁻ 11.95 1702.0 (TACGT)₂ 12.35 1658.6 [M − 2H]²⁻ 12.381658.5 *0.05 M triethylammonium bicarbonate buffer/acetonitrile

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1. A compound represented by the formula:

wherein: R¹ is selected from aryl or alkoxy-substituted aryl, or aryloxyor substituted aryloxy; R² is selected from hydrogen, halogen, alkyl orsubstituted alkyl, alkoxy or substituted alkoxy, aryl or substitutedaryl, aryloxy or substituted aryloxy, amino or substituted amino or anitro group; R³ is selected from hydrogen, alkoxy or substituted alkoxy,aryl or substituted aryl, aryloxy or substituted aryloxy, amino orsubstituted amino, or an unsubstituted or substituted heterocyclicgroup; R⁴ is an alkyl group substituted with one or more halogensubstituents; R⁵ is selected from hydrogen, halogen, alkyl orsubstituted alkyl, alkoxy or substituted alkoxy, aryl or substitutedaryl, aryloxy or substituted aryloxy, amino or substituted amino, anitro group or an unsubstituted or substituted heterocyclic group; and,R⁶ is selected from hydrogen, halogen, alkyl or substituted alkyl,alkoxy or substituted alkoxy, aryl or substituted aryl, aryloxy orsubstituted aryloxy, or amino or substituted amino, or an unsubstitutedor substituted heterocyclic group.
 2. The compound of claim 1, whereinR⁴ is ClCH₂, Cl₂CH, Cl₃C or F₃C.
 3. The compound of claim 1 wherein: R¹is a phenyl or alkoxy substituted phenyl group, a 3-alkoxy substitutedphenyl group; and/or, R² is hydrogen or a substituted or unsubstitutedalkoxy group; and/or, R³ is hydrogen, a substituted or unsubstitutedalkoxy group or an amino or substituted amino group; and/or, R⁴ isClCH₂, CL₂CH, Cl₃C or F₃C; and/or, R⁵ is hydrogen or a nitro group;and/or R⁶ is hydrogen.
 4. The compound of claim 1, wherein: R¹ is C₆H5,m-CH₃O—C₆H₄, or p-CH₃O—C₆H₄; and/or R² is hydrogen or OCH₃; and/or, R³is hydrogen, OCH₃ or N(CH₃)₂; and/or, R⁴ is ClCH₂, Cl₂CH, Cl₃C or F₃C;and/or R⁵ is hydrogen or a nitro group; and/or R⁶ is hydrogen.
 5. Thecompound of claim 1, wherein the compound is trichloroacetic acid(4,5-dimethoxy-2-nitro-phenyl)-phenyl-methyl ester (39); ortrichloroacetic acid (4,5-dimethoxy-2,6-dinitro-phenyl)-phenyl-methylester (40), or trichloroacetic acid(5-dimethylamino-2,6-dinitro-phenyl)-phenyl-methyl ester (43).
 6. Apolymeric film that comprises a compound according to claim
 1. 7. Amethod of synthesizing a nucleic acid molecule or peptide on a solidsupport, the method comprising: (a) bringing a nucleic acid molecule orpeptide having a protected terminus into contact with a compound havinga formula as defined in claim 1 (b) photolysing the compound to producea halogen substituted carboxylic acid capable of removing the protectinggroup from the end of the nucleic acid molecule or peptide; (c)contacting the deprotected nucleic acid molecule or peptide withnucleosides or amino acids, so that the 5′ end of the nucleic acidmolecule or peptide reacts with a nucleoside or amino acid; and (d)repeating steps (a) to (c) until the synthesis of the nucleic acidmolecule or peptide is complete.
 8. The method of claim 7, wherein thehalogen substituted acid generated by the photolysis of the compound iscapable of removing a 5′-O-dimethoxytrityl (DMT) protecting grouppresent on the 5′ end of a nucleic acid molecule or peptide.
 9. Themethod of claim 7 wherein the compounds are used in the synthesis of anarray having a plurality of array elements, wherein a nucleic acidmolecule or peptide is synthesized at each element of the array.
 10. Themethod of claim 7, wherein the nucleic acid molecules or peptides aresynthesised on a glass surface.
 11. The method of claim 7, photolysingthe compounds is carried out by projection lithography.
 12. The methodof claim 7, the compounds are immobilized in a solid polymer film toprevent or reduce acid diffusion from irradiated to non-irradiated arrayelements.
 13. The method of claim 7, wherein a base or a buffer isprovided at each element in the array to neutralise acid generated bystray light photolysing the compound present at elements of the arrayother than those elements targeted for synthesis.
 14. The method ofclaim 7, wherein the compounds are capable of being photolysed byvisible light.
 15. The method of claim 11, wherein the projectionlithography is maskless projection lithography.